Selective Vpac2 Receptor Peptide Agonists

ABSTRACT

The present invention encompasses PEGylated peptides that selectively activate the VPAC2 receptor and are useful in the treatment of diabetes.

The present invention relates to selective VPAC2 receptor peptide agonists.

In particular, the present invention relates to selective VPAC2 receptor peptide agonists which are covalently attached to one or more molecules of polyethylene glycol or a derivative thereof.

Type 2 diabetes, or non-insulin dependent diabetes mellitus (NIDDM), is the most common form of diabetes, affecting 90% of people with diabetes. With NIDDM, patients have impaired β-cell function resulting in insufficient insulin production and/or decreased insulin sensitivity. If NIDDM is not controlled, excess glucose accumulates in the blood, resulting in hyperglycemia. Over time, more serious complications may arise including renal dysfunction, cardiovascular problems, visual loss, lower limb ulceration, neuropathy, and ischemia. Treatments for NIDDM include improving diet, exercise, and weight control as well as using a variety of oral medications. Individuals with NIDDM can initially control their blood glucose levels by talking such oral medications. However, these medications do not slow the progressive loss of β-cell function that occurs in type 2 diabetes patients and, thus, are not sufficient to control blood glucose levels in the later stages of the disease. Also, treatment with currently available medications exposes NIDDM patients to potential side effects such as hypoglycemia, gastrointestinal problems, fluid retention, oedema, and/or weight gain.

Compounds, such as peptides that are selective for a particular G-protein coupled receptor known as the VPAC2 receptor, were initially identified by modifying vasoactive intestinal peptide (VIP) and/or pituitary adenylate cyclase-activating polypeptide (PACAP). (See, for example, Xia et al., J Pharmacol Exp Ther., 281:629-633 (1997); Tsutsumi et al., Diabetes, 51:1453-1460 (2002), WO 01/23420, WO 2004/006839).

PACAP belongs to the secretin/glucagon/vasoactive intestinal peptide (VIP) family of peptides and works through three G-protein-coupled receptors that exert their action through the cAMP-mediated and other Ca²⁺-mediated signal transduction pathways. These receptors are known as the PACAP-preferring type 1 (PAC1) receptor (Isobe, et al., Regul. Pept., 110:213-217 (2003); Ogi, et al., Biochem. Biophys. Res. Commun., 196:1511-1521 (1993)) and the two VIP-shared type 2 receptors (VPAC1 and VPAC2) (Sherwood et al., Endocr. Rev., 21:619-670 (2000); Hammar et al., Pharmacol Rev, 50:265-270 (1998); Couvineau, et al., J. Biol. Chem., 278:24759-24766 (2003); Sreedharan, et al., Biochem. Biophys. Res. Commun., 193:546-553 (1993); Lutz, et al., FEBS Lett., 458: 197-203 (1999); Adamou, et al., Biochem. Biophys. Res. Commun., 209: 385-392 (1995)).

PACAP has comparable activities towards all three receptors, whilst VIP selectively activates the two VPAC receptors (Tsutsumi et al., Diabetes, 51:1453-1460 (2002)). Both VIP (Eriksson et al., Peptides, 10: 481-484 (1989)) and PACAP (Filipsson et al., JCEM, 82:3093-3098 (1997)) have been shown to not only stimulate insulin secretion in man when given intravenously but also increase glucagon secretion and hepatic glucose output. As a consequence, PACAP or VIP stimulation generally does not result in a net improvement of glycemia. Activation of multiple receptors by PACAP or VIP also has broad physiological effects on nervous, endocrine, cardiovascular, reproductive, muscular, and immune systems (Gozes et al., Curr. Med. Chem., 6:1019-1034 (1999)). Furthermore, it appears that VIP-induced watery diarrhoea in rats is mediated by only one of the VPAC receptors, VPAC1 (Ito et al., Peptides, 22:1139-1151 (2001); Tsutsumi et al., Diabetes, 51:1453-1460 (2002)). In addition, the VPAC1 and PAC1 receptors are expressed on α-cells and hepatocytes and, thus, are most likely involved in the effects on hepatic glucose output.

WO 91/06565 (Diacel Chemical Industries and Meiji Seika Kaisha Ltd) describes three peptides having an activity of relaxing smooth or unstriated muscles. Described are peptides which include a helodermin derivative comprising a combination of the amino acid sequence of VIP with a part of the amino acid sequence of helodermin, as well as a peptide composed of a combination of a part of the amino acid sequence of VIP with another part of the amino acid sequence of helodermin.

Known natural VIP related peptides include helodermin and helospectin, which are isolated from the salivary excretions of the Gila Monster (Heloderma Suspectum). The main difference between helodermin and helospectin is the presence in helodermin of two consecutive acidic residues in positions 8 and 9. The different behaviour of helodermin and helospectin in rat and human is of particular interest as lizard peptides are long acting VIP analogues.

Recent studies have shown that peptides selective for the VPAC2 receptor are able to stimulate insulin secretion from the pancreas without gastrointestinal (GI) side effects and without enhancing glucagon release and hepatic glucose output (Tsutsumi et al., Diabetes, 51:1453-1460 (2002)).

Many of the VPAC2 receptor peptide agonists reported to date, however, have less than desirable potency, selectivity, and stability profiles, which could impede their clinical viability. In addition, many of these peptides are not suitable for commercial candidates as a result of stability issues associated with the polypeptides in formulation, as well as issues with the short half-life of these polypeptides in vivo. It has, furthermore, been identified that some VPAC2 receptor peptide agonists are inactivated by dipeptidyl-peptidase (DPP-IV). A short serum half-life could hinder the use of these agonists as therapeutic agents. There is, therefore, a need for new therapies, which overcome the problems associated with current medications for NIDDM.

The present invention seeks to provide improved compounds that are selective for the VPAC2 receptor and which induce insulin secretion from the pancreas only in the presence of high blood glucose levels. The compounds of the present invention are peptides, which are believed to also improve beta cell function. These peptides can have the physiological effect of inducing insulin secretion without GI side effects or a corresponding increase in hepatic glucose output and also generally have enhanced selectivity, potency, and/or in vivo stability of the peptide compared to known VPAC2 receptor peptide agonists.

The present invention also seeks to provide selective VPAC2 receptor peptide agonists, which have reduced clearance and improved in vivo stability. It is desirable that the agonists of the present invention be administered a minimum number of times during a prolonged period of time.

According to a first aspect of the present invention, there is provided a PEGylated VPAC2 receptor peptide agonist comprising a sequence of the formula:

Formula 7 (SEQ ID NO:12) Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Thr-Xaa₈-Xaa₉-Xaa₁₀- Thr-Xaa₁₂-Xaa₁₃-Xaa₁₄-Xaa₁₅-Xaa₁₆-Xaa₁₇-Xaa₁₈- Xaa₁₉-Xaa₂₀-Xaa₂₁-Xaa₂₂-Xaa₂₃-Xaa₂₄-Xaa₂₅-Xaa₂₆- Xaa₂₇-Xaa₂₈-Xaa₂₉-Xaa₃₀-Xaa₃₁-Xaa₃₂-Xaa₃₃-Xaa₃₄- Xaa₃₅-Xaa₃₆-Xaa₃₇-Xaa₃₈-Xaa₃₉-Xaa₄₀ wherein:

-   Xaa₁ is: His, dH, or is absent; -   Xaa₂ is: dA, Ser, Val, Gly, Thr, Leu, dS, Pro, or Aib; -   Xaa₃ is: Asp or Glu; -   Xaa₄ is: Ala, Ile, Tyr, Phe, Val, Thr, Leu, Trp, Gly, dA, Aib, or     NMeA; -   Xaa₅ is: Val, Leu, Phe, Ile, Thr, Trp, Tyr, dV, Aib, or NMeA; -   Xaa₆ is: Phe, Ile, Leu, Thr, Val, Trp, or Tyr; -   Xaa₈ is: Asp, Glu, Ala, Lys, Leu, Arg, or Tyr; -   Xaa₉ is: Asn, Gln, Asp, Glu, Ser, Cys, Lys, or K(CO(CH₂)₂SH); -   Xaa₁₀ is: Tyr, Trp, Tyr(OMe), Ser, Cys, or Lys; -   Xaa₁₂ is: Arg, Lys, Glu, hR, Orn, Lys (isopropyl), Aib, Cit, Ala,     Leu, Gln, Phe, Ser, or Cys; -   Xaa₁₃ is: Leu, Phe, Glu, Ala, Aib, Ser, Cys, Lys or K(CO(CH₂)₂SH); -   Xaa₁₄ is: Arg, Leu, Lys, Ala, hR, Orn, Lys (isopropyl), Phe, Gln,     Aib, Cit, Ser, or Cys; -   Xaa₁₅ is: Lys, Ala, Arg, Glu, Leu, hR, Orn, Lys (isopropyl), Phe,     Gln, Aib, K(Ac), Cit, Ser, Cys, K(W), or K(CO(CH₂)₂SH); -   Xaa₁₆ is: Gln, Lys, Glu, Ala, hR, Orn, Lys (isopropyl), Cit, Ser,     Cys, K(CO(CH₂)₂SH), or K(W); -   Xaa₁₇ is: Val, Ala, Leu, Ile, Met, Nle, Lys, Aib, Ser, Cys,     K(CO(CH₂)₂SH), or K(W); -   Xaa₁₈ is: Ala, Ser, Cys, Lys, K(CO(CH₂)₂SH), or K(W); -   Xaa₁₉ is: Val, Ala, Glu, Phe, Gly, H is, Ile, Lys, Leu, Met, Asn,     Pro, Gln, Arg, Ser, Thr, Trp, Tyr, Cys, Asp, K(CO(CH₂)₂SH), or K(W); -   Xaa₂₀ is: Lys, Gln, hR, Arg, Ser, His, Orn, Lys (isopropyl), Ala,     Aib, Trp, Thr, Leu, Ile, Phe, Tyr, Val, K(Ac), Cit, Cys,     K(CO(CH₂)₂SH), or K(W); -   Xaa₂₁ is: Lys, His, Arg, Ala, Phe, Aib, Leu, Gln, Orn, hR, K(Ac),     Cit, Ser, Cys, Val, Tyr, Ile, Thr, Trp, K(W), or K(CO(CH₂)₂SH); -   Xaa₂₂ is: Tyr, Trp, Phe, Thr, Leu, Ile, Val, Tyr(OMe), Ala, Aib,     Ser, Cys, Lys, K(W), or K(CO(CH₂)₂SH); -   Xaa₂₃ is: Leu, Phe, Ile, Ala, Trp, Thr, Val, Aib, Ser, Cys, Lys,     K(W), or K(CO(CH₂)₂SH); -   Xaa₂₄ is: Gln, Glu, Asn, Ser, Cys, Lys, K(CO(CH₂)₂SH), or K(W); -   Xaa₂₅ is: Ser, Asp, Phe, Ile, Leu, Thr, Val, Trp, Gln, Asn, Tyr,     Aib, Glu, Cys, Lys, K(CO(CH₂)₂SH), or K(W); -   Xaa₂₆ is: Ile, Leu, Thr, Val, Trp, Tyr, Phe, Aib, Ser, Cys, Lys,     K(CO(CH₂)₂SH), or K(W); -   Xaa₂₇ is: Lys, hR, Arg, Gln, Ala, Asp, Glu, Phe, Gly, His, Ile, Met,     Asn, Pro, Ser, Thr, Val, Trp, Tyr, Lys (isopropyl), Cys, Leu, Orn,     dK, K(W), or K(CO(CH₂)₂SH); -   Xaa₂₈ is: Asn, Asp, Gln, Lys, Arg, Aib, Orn, hR, Cit, Pro, dK, Ser,     Cys, K(CO(CH₂)₂SH), K(W), or is absent; -   Xaa₂₉ is: Lys, Ser, Arg, Asn, hR, Ala, Asp, Glu, Phe, Gly, His, Ile,     Leu, Met, Pro, Gln, Thr, Val, Trp, Tyr, Cys, Orn, Cit, Aib, K(W),     K(CO(CH₂)₂SH), or is absent; -   Xaa₃₀ is: Arg, Lys, Ile, Ala, Asp, Glu, Phe, Gly, His, Leu, Met,     Asn, Pro, Gln, Ser, Thr, Val, Trp, Tyr, Cys, hR, Cit, Aib, Orn,     K(W), K(CO(CH₂)₂SH), or is absent; -   Xaa₃₁ is: Tyr, His, Phe, Thr, Cys, Ser, Lys, Gln, K(W),     K(CO(CH₂)₂SH), or is absent; -   Xaa₃₂ is: Ser, Cys, Lys, or is absent; -   Xaa₃₃ is: Trp, or is absent; -   Xaa₃₄ is: Cys or is absent; -   Xaa₃₅ is: Glu or is absent; -   Xaa₃₆ is: Pro or is absent; -   Xaa₃₇ is: Gly or is absent; -   Xaa₃₈ is: Trp or is absent; -   Xaa₃₉ is: Cys or is absent; and -   Xaa₄₀ is: Arg or is absent

provided that if Xaa₂₈, Xaa₂₉, Xaa₃₀, Xaa₃₁, Xaa₃₂, Xaa₃₃, Xaa₃₄, Xaa₃₅, Xaa₃₆, Xaa_(37 ,)Xaa_(38 ,)or Xaa₃₉ is absent, the next amino acid present downstream is the next amino acid in the peptide agonist sequence,

and a C-terminal extension wherein the N-terminus of the C-terminal extension is linked to the C-terminus of the peptide of Formula 7 and wherein the C-terminal extension comprises an amino acid sequence of the formula:

Formula 15 (SEQ ID NO: 20) Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa₁₀ wherein:

-   Xaa₁ is: Ser, Cys, Lys, K(W), K(CO(CH₂)₂SH), or absent; -   Xaa₂ is: Arg, Ser, hR, Orn, His, Cys, Lys, K(W), K(CO(CH₂)₂SH), or     absent; -   Xaa₃ is: Thr, Cys, Lys, K(W), K(CO(CH₂)₂SH), or absent; -   Xaa₄ is: Ser, Cys, Lys, K(W), K(CO(CH₂)₂SH), or absent; -   Xaa₅ is: Pro, Ser, Ala, Cys, Lys, K(W), K(CO(CH₂)₂SH), or absent; -   Xaa₆ is: Pro, Ser, Ala, Arg, Cys, Lys, K(W), K(CO(CH₂)₂SH), or     absent; -   Xaa₇ is: Pro, Ser, Ala, Cys, Lys, K(W), K(CO(CH₂)₂SH), or absent; -   Xaa₈ is: Lys, K(W), Pro, Cys, K(CO(CH₂)₂SH), or absent; -   Xaa₉ is: K(E-C₁₆), Ser, Cys, Lys, K(W), K(CO(CH₂)₂SH), or absent;     and -   Xaa₁₀ is: Ser, Cys, Lys, K(W), K(CO(CH₂)₂SH), or absent;

provided that at least four of Xaa₁ to Xaa₁₀ of the C-terminal extension are present and provided that if Xaa₁, Xaa₂, Xaa₃, Xaa₄, Xaa₅, Xaa₆, Xaa₇, Xaa₈, or Xaa₉ is absent, the next amino acid present downstream is the next amino acid in the C-terminal extension and wherein the C-terminal amino acid may be amidated,

and wherein;

at least one of the Cys residues in the peptide agonist is covalently attached to a PEG molecule, or

at least one of the Lys residues in the peptide agonist is covalently attached to a PEG molecule, or

at least one of the K(W) in the peptide agonist is covalently attached to a PEG molecule, or

at least one of the K(CO(CH₂)₂SH) in the peptide agonist is covalently attached to a PEG molecule, or

the carboxy-terminal amino acid of the peptide agonist is covalently attached to a PEG molecule, or a combination thereof.

Preferably, at least five of Xaa₁ to Xaa₁₀ of the C-terminal extension of Formula 15 are present. More preferably at least six, seven, eight, nine or all of Xaa₁ to Xaa₁₀ are present.

It is preferable that the C-terminal extension has no more than three of any one of the following; Cys, Lys, K(W) or K(CO(CH₂)₂SH). It is more preferable that the C-terminal extension has no more than two of any of these residues. It is even more preferable that the C-terminal extension has no more than one of any of these residues.

Preferably, the PEGylated VPAC2 receptor peptide agonist comprises a sequence of the formula:

Formula 9 (SEQ ID NO: 14) Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Thr-Xaa₈-Xaa₉-Xaa₁₀- Thr-Xaa₁₂-Xaa₁₃-Xaa₁₄-Xaa₁₅-Xaa₁₆-Xaa₁₇-Xaa₁₈- Xaa₁₉-Xaa₂₀-Xaa₂₁-Xaa₂₂-Xaa₂₃-Xaa₂₄-Xaa₂₅-Xaa₂₆- Xaa₂₇-Xaa₂₈-Xaa₂₉-Xaa₃₀-Xaa₃₁-Xaa₃₂ wherein:

-   Xaa₁ is: His, dH, or is absent; -   Xaa₂ is: dA, Ser, Val, Gly, Thr, Leu, dS, Pro, or Aib; -   Xaa₃ is: Asp or Glu; -   Xaa₄ is: Ala, Ile, Tyr, Phe, Val, Thr, Leu, Trp, Gly, dA, Aib, or     NMeA; -   Xaa₅ is: Val, Leu, Phe, Ile, Thr, Trp, Tyr, dV, Aib, or NMeA; -   Xaa₆ is: Phe, Ile, Leu, Thr, Val, Trp, or Tyr; -   Xaa₈ is: Asp, Glu, Ala, Lys, Leu, Arg, or Tyr; -   Xaa₉ is: Asn, Gln, Glu, Ser, Cys, or Lys; -   Xaa₁₀ is: Tyr, Trp, Tyr(OMe), Ser, Cys, or Lys; -   Xaa₁₂ is: Arg, Lys, hR, Orn, Aib, Cit, Ala, Leu, Gln, Phe, Ser, or     Cys; -   Xaa₁₃ is: Leu, Phe, Glu, Ala, Aib, Ser, Cys, Lys, or K(CO(CH₂)₂SH); -   Xaa₁₄ is: Arg, Leu, Lys, Ala, hR, Orn, Phe, Gln, Aib, Cit, Ser, or     Cys; -   Xaa₁₅ is: Lys, Ala, Arg, Glu, Leu, hR, Orn, Phe, Gln, Aib, K(Ac),     Cit, Ser, Cys, or K(W); -   Xaa₁₆ is: Gln, Lys, Ala, hR, Orn, Cit, Ser, Cys, or K(CO(CH₂)₂SH); -   Xaa₁₇ is: Val, Ala, Leu, Ile, Met, Nle, Lys, Aib, Ser, Cys, or     K(CO(CH₂)₂SH); -   Xaa₁₈ is: Ala, Ser, Cys, or Lys; -   Xaa₁₉ is: Ala, Gly, Leu, Ser, Cys, Lys, or K(CO(CH₂)₂SH); Cit, or     Cys; -   Xaa₂₀ Lys, Gln, hR, Arg, Ser, Orn, Ala, Aib, Trp, Thr, Leu, Ile,     Phe, Tyr, Val, K(Ac), Cit, or Cys; -   Xaa₂₁ is: Lys, Arg, Ala, Phe, Aib, Leu, Gln, Orn, hR, K(Ac), Cit,     Ser, or Cys; -   Xaa₂₂ is: Tyr, Trp, Phe, Thr, Leu, Ile, Val, Tyr(OMe), Ala, Aib,     Ser, Cys, or Lys; -   Xaa₂₃ is: Leu, Phe, Ile, Ala, Trp, Thr, Val, Aib, Ser, Cys, or Lys; -   Xaa₂₄ is: Gln, Asn, Ser, Cys, Lys, or K(CO(CH₂)₂SH); -   Xaa₂₅ is: Ser, Asp, Phe, Ile, Leu, Thr, Val, Trp, Gln, Asn, Tyr,     Aib, Glu, Cys, Lys, or K(CO(CH₂)₂SH); -   Xaa₂₆ is: Ile, Leu, Thr, Val, Trp, Tyr, Phe, Aib, Ser, Cys, Lys, or     K(CO(CH₂)₂SH); -   Xaa₂₇ is: Lys, hR, Arg, Gln, Orn, dK, Ser, or Cys; -   Xaa₂₈ is: Asn, Gln, Lys, Arg, Aib, Orn, hR, Cit, Pro, dK, Ser, Cys,     K(CO(CH₂)₂SH), or is absent; -   Xaa₂₉ is: Lys, Ser, Arg, Asn, hR, Orn, Cit, Aib, Cys, or is absent; -   Xaa₃₀ is: Arg, Lys, Ile, hR, Cit, Aib, Orn, Ser, Cys, or is absent; -   Xaa₃₁ is: Tyr, His, Phe, Lys, Ser, Cys, Gln, or is absent; and -   Xaa₃₂ is: Cys, Ser, Lys, or is absent;

provided that if Xaa₂₈, Xaa₂₉, Xaa₃₀, or Xaa₃₁ is absent, the next amino acid present downstream is the next amino acid in the peptide agonist sequence,

and a C-terminal extension wherein the N-terminus of the C-terminal extension is linked to the C-terminus of the peptide of Formula 9 and wherein the C-terminal extension comprises an amino acid sequence of the formula:

Formula 8 (SEQ ID NO: 13) Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa₁₀ wherein:

Xaa₁ is: Ser, Cys, Lys, or absent;

Xaa₂ is: Arg, Ser, hR, Orn, His, Cys, Lys, or absent;

Xaa₃ is: Thr, Cys, Lys, or absent;

Xaa₄ is: Ser, Cys, Lys, or absent;

Xaa₅ is: Pro, Ser, Ala, Cys, Lys, or absent;

Xaa₆ is: Pro, Ser, Ala, Arg, Cys, Lys, or absent;

Xaa₇ is: Pro, Ser, Ala, Cys, Lys, or absent;

Xaa₈ is: Lys, K(W), Pro, Cys, or absent;

Xaa₉ is: K(E-C₁₆), Ser, Cys, Lys, or absent; and

Xaa₁₀ is: Ser, Cys, Lys, or absent;

provided that at least four of Xaa₁ to Xaa₁₀ of the C-terminal extension are present and provided that if Xaa₁, Xaa₂, Xaa₃, Xaa₄, Xaa₅, Xaa₆, Xaa₇, Xaa₈, or Xaa₉ is absent, the next amino acid present downstream is the next amino acid in the C-terminal extension and wherein the C-terminal amino acid may be amidated,

and wherein;

at least one of the Cys residues in the peptide agonist is covalently attached to a PEG molecule, or

at least one of the Lys residues in the peptide agonist is covalently attached to a PEG molecule, or

at least one of the K(CO(CH₂)₂SH) in the peptide agonist is covalently attached to a PEG molecule, or

at least one of the K(W) in the peptide agonist is covalently attached to a PEG molecule, or

the carboxy-terminal amino acid is covalently attached to a PEG molecule, or a combination thereof.

Preferably, at least five of Xaa₁ to Xaa₁₀ of the C-terminal extension of Formula 8 are present. More preferably at least six, seven, eight, nine or all of Xaa₁ to Xaa₁₀ are present.

Preferably, Xaa₃₀, and Xaa₃₁ of Formula 7 (SEQ ID NO: 12) or Formula 9 (SEQ ID NO: 14) are absent. Alternatively, Xaa₂₉, Xaa₃₀, and Xaa₃₁ of Formula 7 (SEQ ID NO: 12) or Formula 9 (SEQ ID NO: 14) are all absent.

The PEGylated VPAC2 receptor peptide agonist preferably comprises a sequence of the formula:

Formula 10 (SEQ ID NO: 15) His-Ser-Xaa₃-Ala-Val-Phe-Thr-Xaa₈-Xaa₉-Xaa₁₀-Thr- Xaa₁₂-Xaa₁₃-Xaa₁₄-Xaa₁₅-Xaa₁₆-Xaa₁₇-Xaa₁₈-Xaa₁₉- Xaa₂₀-Xaa₂₁-Xaa₂₂-Xaa₂₃-Xaa₂₄-Xaa₂₅-Xaa₂₆-Xaa₂₇- Xaa₂₈ wherein:

Xaa₃ is: Asp, or Glu;

Xaa₈ is: Asp, or Glu;

Xaa₉ is: Asn, Gln, Ser, Cys, or Lys;

Xaa₁₀ is: Tyr, or Tyr(OMe);

Xaa₁₂ is: Arg, hR, Lys, Orn, Ser, or Cys;

Xaa₁₃ is: Leu, Ser, Cys, or Lys;

Xaa₁₄ is: Arg, Leu, Gln, Aib, hR, Orn, Cit, Lys, Ala, Ser, or Cys;

Xaa₁₅ is: Lys, Leu, Ala, Aib, Orn, Ser, Cys, or Arg;

Xaa₁₆ is: Gln, Lys, Ala, Ser, or Cys;

Xaa₁₇ is: Val, Ala, Leu, Ile, Lys, Nle, Ser, or Cys;

Xaa₁₈ is: Ala, Ser, Cys, or Lys;

Xaa₁₉ is: Ala, Ser, Cys, or Lys;

Xaa₂₀ is: Lys, Aib, Val, Leu, Ala, Gln, Ser, Cys, or Arg;

Xaa₂₁ is: Lys, Aib, Orn, Ala, Gln, Ser, Cys, or Arg;

Xaa₂₂ is: Tyr, Ser, Cys, or Lys;

Xaa₂₃ is: Leu, Ser, Cys, or Lys;

Xaa₂₄ is: Gln, Ser, Cys, or Lys;

Xaa₂₅ is: Ser, Cys, or Lys;

Xaa₂₆ is: Ile, Ser, Cys, or Lys;

Xaa₂₇ is: Lys, Orn, hR, Ser, Cys, or Arg; and

Xaa₂₈ is: Asn, Gln, Lys, hR, Aib, Pro, Orn, Ser, or Cys;

and a C-terminal extension wherein the N-terminus of the C-terminal extension is linked to the C-terminus of the peptide of Formula 10 and wherein the C-terminal extension comprises an amino acid sequence of the formula:

Formula 8 (SEQ ID NO: 13) Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa₁₀ wherein:

Xaa₁ is: Ser, Cys, Lys, or absent;

Xaa₂ is: Arg, Ser, hR, Orn, His, Cys, Lys, or absent;

Xaa₃ is: Thr, Cys, Lys, or absent;

Xaa₄ is: Ser, Cys, Lys, or absent;

Xaa₅ is: Pro, Ser, Ala, Cys, Lys, or absent;

Xaa₆ is: Pro, Ser, Ala, Arg, Cys, Lys, or absent;

Xaa₇ is: Pro, Ser, Ala, Cys, Lys, or absent;

Xaa₈ is: Lys, K(W), Pro, Cys or absent;

Xaa₉ is: K(E-C₁₆), Ser, Cys, Lys or absent; and

Xaa₁₀ is: Ser, Cys, Lys, or absent;

provided that at least four of Xaa₁ to Xaa₁₀ of the C-terminal extension are present and provided that if Xaa₁, Xaa₂, Xaa₃, Xaa₄, Xaa₅, Xaa₆, Xaa₇, Xaa₈, or Xaa₉ is absent, the next amino acid present downstream is the next amino acid in the C-terminal extension and wherein the C-terminal amino acid may be amidated,

and wherein;

at least one of the Cys residues in the peptide agonist is covalently attached to a PEG molecule, or

at least one of the Lys residues in the peptide agonist is covalently attached to a PEG molecule, or

the K(W) in the peptide agonist is covalently attached to a PEG molecule, or

the carboxy-terminal amino acid is covalently attached to a PEG molecule, or a combination thereof.

Preferably, the PEGylated VPAC2 receptor peptide agonist of the present invention comprises a sequence of the Formula 7 (SEQ ID NO: 12), Formula 9 (SEQ ID NO: 14) or Formula 10 (SEQ ID NO: 15) wherein Xaa₃ is Asp or Glu, Xaa₈ is Asp or Glu, Xaa₉ is Asn or Gln, Xaa₁₂ is Arg, hR, Lys, or Orn, Xaa₁₄ is Arg, Gln, Aib, hR, Orn, Cit, Lys, Ala, or Leu, Xaa₁₅ is Lys, Leu, Aib, Orn, or Arg, Xaa₁₆ is Gln or Lys, Xaa₁₇ is Val, Leu, Ala, Ile, Lys or Nle, Xaa₂₀ is Lys, Val, Leu, Aib, Ala, Gln, or Arg, Xaa₂₁ is Lys, Aib, Orn, Ala, Gln, or Arg, Xaa₂₇ is Lys, Orn, hR or Arg, and Xaa₂₈ is Asn, Gln, Lys, hR, Aib, Pro, or Orn.

More preferably, the PEGylated VPAC2 receptor peptide agonist of the present invention comprises a sequence of the Formula 7 (SEQ ID NO: 12), Formula 9 (SEQ ID NO: 14) or Formula 10 (SEQ ID NO: 15) wherein Xaa₁₂ is Arg, hR, or Orn, Xaa₁₄ is Arg, Aib, Gln, Ala, Leu, Lys, or Orn, Xaa₁₅ is Lys or Aib, Xaa₁₇ is Val or Leu, Xaa₂₀ is Lys or Aib, Xaa₂₁ is Lys, Aib, or Gln and Xaa₂₈ is Asn or Gln.

Preferably, the PEGylated VPAC2 receptor peptide agonist of the present invention has the Formula 7 (SEQ ID NO: 12), Formula 9 (SEQ ID NO: 14) or Formula 10 (SEQ ID NO: 15) wherein either Xaa₁₄ or Xaa₁₅ is Aib.

Preferably, the PEGylated VPAC2 receptor peptide agonist of the present invention has the Formula 7 (SEQ ID NO: 12); Formula 9 (SEQ ID: 14) or Formula 10 (SEQ ID NO: 15) wherein either Xaa₂₀ or Xaa₂₁ is Aib.

More preferably, the PEGylated VPAC2 receptor peptide agonist of the present invention has the Formula 7 (SEQ ID NO: 12), Formula 9 (SEQ ID NO: 14) or Formula 10 (SEQ ID NO: 15) wherein either Xaa₁₄ or Xaa₁₅ is Aib and either Xaa₂₀ or Xaa₂₁ is Aib. It is especially preferred that Xaa₁₅ is Aib and Xaa₂₀ is Aib.

Preferably, the VPAC2 receptor peptide agonist of the present invention comprises a sequence of the Formula 7 (SEQ ID NO. 12), Formula 9 (SEQ ID NO 14) or Formula 10 (SEQ ID NO 15) wherein Xaa₁₅ is Aib, Xaa₂₀ is Aib, and Xaa₁₂, Xaa₂₁, Xaa₂₇ and Xaa₂₈ are all Orn. More preferably, Xaa₁₅ is Aib, Xaa₂₀ is Aib, Xaa₁₂, Xaa₂₁, Xaa₂₇ and Xaa₂₈ are all Orn, Xaa₈ is Glu, Xaa₉ is Gln and Xaa₁₀ is Tyr(OMe). Alternatively, any one or more of Xaa₈, Xaa₉, Xaa₁₀, Xaa₁₂, Xaa₁₅, Xaa₂₀, Xaa₂₁, Xaa₂₇ and Xaa₂₈ may be a PEGylated Lys, Cys, K(CO(CH₂)₂SH) or K(W), whilst all the other positions have the preferred amino acid substitutions as described.

Preferably, the PEGylated VPAC2 receptor peptide agonist of the present invention has the Formula 7 (SEQ ID NO: 12), Formula 9 (SEQ ID NO: 14) or Formula 10 (SEQ ID NO: 15) wherein Xaa₂₈ is Gln.

Preferably, the PEGylated VPAC2 receptor peptide agonist of the present invention has the Formula 7 (SEQ ID NO: 12), Formula 9 (SEQ ID NO: 14) or Formula 10 (SEQ ID NO: 15) wherein Xaa₁₂ is hR or Orn and Xaa₂₇ is hR or Orn.

More preferably, the PEGylated VPAC2 receptor peptide agonist of the invention comprises a sequence of the formula:

Formula 11 (SEQ ID NO: 16) His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Xaa₉-Tyr-Thr-Arg- Leu-Xaa₁₄-Xaa₁₅-Xaa₁₆-Xaa₁₇-Ala-Ala-Xaa₂₀-Lys-Tyr- Leu-Gln-Ser-Ile-Lys-Xaa₂₈ wherein:

Xaa₉ is: Asn, or Gln;

Xaa₁₄ is: Arg, or Leu;

Xaa₁₅ is: Lys, Leu, or Aib;

Xaa₁₆ is: Gln, Lys, or Ala;

Xaa₁₇ is: Val, or Ala;

Xaa₂₀ is: Lys, or Aib; and

Xaa₂₈ is: Asn, or Gln;

and a C-terminal extension wherein the N-terminus of the C-terminal extension is linked to the C-terminus of the peptide of Formula 11 and wherein the C-terminal extension comprises an amino acid sequence of the formula:

Formula 8 (SEQ ID NO:13) Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa₁₀ wherein:

Xaa₁ is: Ser, Cys, Lys or absent;

Xaa₂ is: Arg, Ser, hR, Orn, His, Cys, Lys or absent;

Xaa₃ is: Thr, Cys, Lys, or absent;

Xaa₄ is: Ser, Cys, Lys, or absent;

Xaa₅ is: Pro, Ser, Ala, Cys, Lys, or absent;

Xaa₆ is: Pro, Ser, Ala, Arg, Cys, Lys, or absent;

Xaa₇ is: Pro, Ser, Ala, Cys, Lys, or absent;

Xaa₈ is: Lys, K(W), Pro, Cys, or absent;

Xaa₉ is: K(E-C₁₆), Ser, Cys, Lys, or absent; and

Xaa₁₀ is: Ser, Cys, Lys or absent;

provided that at least four of Xaa₁ to Xaa₁₀ of the C-terminal extension are present and provided that if Xaa₁, Xaa₂, Xaa₃, Xaa₄, Xaa₅, Xaa₆, Xaa₇, Xaa₈, or Xaa₉ is absent, the next amino acid present downstream is the next amino acid in the C-terminal extension and wherein the C-terminal amino acid may be amidated,

and wherein;

at least one of the Cys residues in the peptide agonist is covalently attached to a PEG molecule, or

at least one of the Lys residues in the peptide agonist is covalently attached to a PEG molecule, or

the K(W) in the peptide agonist is covalently attached to a PEG molecule, or

the carboxy-terminal amino acid of the PEG molecule is covalently attached to a PEG molecule, or

a combination thereof.

Preferably, the C-terminal extension of the PEGylated VPAC2 receptor peptide agonist comprises an amino acid sequence of the formula;

Formula 12 (SEQ ID NO: 17) Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉ wherein:

Xaa₁ is: Ser or absent;

Xaa₂ is: Arg, or absent;

Xaa₃ is: Thr or absent;

Xaa₄ is: Ser or absent;

Xaa₅ is: Pro or absent;

Xaa₆ is: Pro or absent;

Xaa₇ is: Pro or absent;

Xaa₈ is: Lys, K(W), Cys, or absent; and

Xaa₉ is: K(E-C₁₆) or absent;

provided that at least four of Xaa₁ to Xaa₉ of the C-terminal extension are present and provided that if Xaa₁, Xaa₂, Xaa₃, Xaa₄, Xaa₅, Xaa₆, Xaa₇, or Xaa₈ is absent, the next amino acid present downstream is the next amino acid in the C-terminal extension and wherein the C-terminal amino acid may be amidated.

Preferably, at least five of Xaa₁ to Xaa₈ of the C-terminal extension are present. More preferably, at least six, seven, eight, or all of Xaa₁ to Xaa₈ are present.

More preferably, the C-terminal extension of the PEGylated VPAC2 receptor peptide agonist is selected from:

SEQ ID NO: 9 SRTSPPP SEQ ID NO: 10 SRTSPPP-NH₂ SEQ ID NO: 21 SSTSPRPPSS SEQ ID NO: 22 SSTSPRPPSS-NH₂ SEQ ID NO: 23 SRTSPPPK(W) SEQ ID NO: 24 SRTSPPPK(W)-NH₂ SEQ ID NO: 25 SRTSPPPC SEQ ID NO: 26 SRTSPPPC-NH₂

The PEG molecule(s) may be covalently attached to any Lys, Cys, K(W), or K(CO(CH₂)₂SH) residues at any position in the peptide agonist. In particular, the PEG molecule(s) may be covalently attached to any Lys, Cys, K(W), or K(CO(CH₂)₂SH) residue at positions 9, 13, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26 and/or 28 of Formula 7, 9, 10, or 11. Alternatively, the PEG molecule(s) may be covalently attached to a residue in the C-terminal extension.

Preferably, there is at least one PEG molecule covalently attached to Xaa₂₅ or any subsequent residue in Formula 7, 9, 10, or 11.

Preferably, there is at least one PEG molecule covalently attached to a residue in the C-terminal extension of the VPAC2 receptor peptide agonist.

Any Lys residue in the VPAC2 receptor peptide agonist may be substituted for a K(W) or K(CO(CH₂)₂SH), which may be PEGylated. In addition, any Cys residue in the peptide agonist may be substituted for a modified cysteine residue, for example, hC. The modified Cys residue may be covalently attached to a PEG molecule.

It is preferred that two of the Cys residues are each covalently attached to a PEG molecule or two of the Lys residues are each covalently attached to a PEG molecule. Alternatively, one of the Cys residues may be covalently attached to a PEG molecule or one of the Lys residues may be covalently attached to a PEG molecule. It is also preferred that the K(W) in the C-terminal extension of the peptide agonist is covalently attached to a PEG molecule.

It is preferred that there is a K(CO(CH₂)₂SH) is the VPAC2 receptor peptide agonist and that this is PEGylated.

Where there is more than one PEG molecule, there may be a combination of Lys, Cys, K(CO(CH₂)₂SH), K(W) and carboxy-terminal amino acid PEGylation. For example, if there are two PEG molecules, one may be attached to a Lys residue and one may be attached to a Cys residue.

Preferably, the PEG molecule is branched. Alternatively, the PEG molecule may be linear.

Preferably, the PEG molecule is between 1,000 daltons and 100,000 daltons in molecular weight. More preferably the PEG molecule is selected from 10,000, 20,000, 30,000, 40,000, 50,000 and 60,000 daltons. Even more preferably, it is selected from 20,000, 40,000, or 60,000. Where there are two PEG molecules covalently attached to the peptide agonist of the present invention, each is 1,000 to 40,000 daltons and preferably, they have molecular weights of 20,000 and 20,000 daltons, 10,000 and 30,000 daltons, 30,000 and 30,000 daltons, or 20,000 and 40,000 daltons.

The VPAC2 receptor peptide agonist sequence may further comprise a histidine residue at the N-terminal region of the peptide sequence before Xaa₁.

Preferably, the VPAC2 receptor peptide agonist of the present invention further comprises a N-terminal modification at the N-terminus of the peptide agonist wherein the N-terminal modification is selected from:

-   -   a) addition of D-histidine, isoleucine, methionine, or         norleucine;     -   b) addition of a peptide comprising the sequence         Ser-Trp-Cys-Glu-Pro-Gly-Trp-Cys-Arg (SEQ ID NO: 24) wherein the         Arg is linked to the N-terminus of the peptide agonist;     -   c) addition of C₁-C₁₆ alkyl optionally substituted with one or         more substituents independently selected from aryl, C₁-C₆         alkoxy, —NH₂, —OH, halogen and —CF₃;     -   d) addition of —C(O)R¹ wherein R¹ is a C₁-C₁₆ alkyl optionally         substituted with one or more substituents independently selected         from aryl, C₁-C₆ alkoxy, —NH₂, —OH, halogen, —SH and —CF₃; an         aryl or aryl C₁-C₄ alkyl optionally substituted with one or more         substituents independently selected from C₁-C₆ alkyl, C₂-C₆         alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkoxy, —NH₂, —OH, halogen and         —CF₃; —NR²R³ wherein R² and R³ are independently hydrogen, C₁-C₆         alkyl, aryl or aryl C₁-C₄ alkyl; —OR⁴ wherein R⁴ is C₁-C₁₆ alkyl         optionally substituted with one or more substituents         independently selected from aryl, C₁-C₆ alkoxy, —NH₂, —OH,         halogen and —CF₃, aryl or aryl C₁-C₄ alkyl optionally         substituted with one or more substituents independently selected         from C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkoxy,         —NH₂, —OH, halogen and —CF₃; Or 5-pyrrolidin-2-one;     -   e) addition of —SO₂R⁵ wherein R⁵ is aryl, aryl C₁-C₄ alkyl or         C₁-C₁₆ alkyl;     -   f) formation of a succinimide group optionally substituted with         C₁-C₆ alkyl or —SR⁶, wherein R⁶ is hydrogen or C₁-C₆ alkyl;     -   g) addition of methionine sulfoxide;     -   h) addition of biotinyl-6-aminohexanoic acid (b-aminocaproic         acid); and     -   i) addition of —C(═NH)—NH₂.

Preferably, the N-terminal modification is the addition of a group selected from: acetyl, propionyl, butyryl, pentanoyl, hexanoyl, methionine, methionine sulfoxide, 3-phenylpropionyl, phenylacetyl, benzoyl, norleucine, D-histidine, isoleucine, 3-mercaptopropionyl, biotinyl-6-aminohexanoic acid, and —C(═NH)—NH₂, and more preferably is the addition of acetyl, hexanoyl, propionyl, 3-phenylpropionyl, and benzoyl.

It will be appreciated by the person skilled in the art that PEGylated VPAC2 receptor peptide agonists comprising various combinations of peptide sequence according to Formula 7, 9, 10 or 11, C-terminal extensions and N-terminal modifications as described herein, may be made based on the above disclosure.

According to a second aspect of the invention, the preferred PEGylated VPAC2 receptor peptide agonists comprise an amino acid sequence selected from the following,

wherein:

at least one of the Lys residues is covalently attached to a PEG molecule, or

the K(W) is covalently attached to a PEG molecule, or

a combination thereof;

SEQ Agonist ID # NO Peptide P5 28 HSDAVFTDNYTRLRKQVAAKKYLQSIKNSRTSPPP-NH₂ P30 29 C6-HSDAVFTDNYTRLRKQVAAKKYLQSIKNSRTSPPP- NH₂ P32 30 Ac-HSDAVFTDNYTRLRKQVAAKKYLQSIKNSRTSPPP- NH₂ P80 31 HSDAVFTDNYTRLRKQVAAKKYLQSIKNSRTSPPPK(E- C16)-NH₂ P81 32 C6-HSDAVFTDNYTRLRKQVAAKKYLQSIKNSRTSPPPK (E-C16)-NH₂ P90 33 3-phenylpropionyl HSDAVFTDNYTRLRKQVAAKKYLQSIKNSRTSPPP-NH₂ P91 34 Benzoyl-HSDAVFTDNYTRLRKQVAAKKYLQSIKNSRT SPPP-NH₂ P95 35 C3-HSDAVFTDNYTRLRKQVAAKKYLQSIKNSRTSPPPK (E-C16)-NH₂ P96 36 HSDAVFTDNYTRLRKQAAAKKYLQSIKNSRTSPPP-NH₂ P97 37 HSDAVFTDNYTRLRKAAAAKKYLQSIKNSRTSPPP-NH₂ P118 38 C6-HSDAVFTDNYTRLRKQVAAKKYLQSIKNSRTSPPPK (W)-NH₂ P128 39 HSDAVFTDNYTRLRKQVAAKKYLQSIKNSRTSPPPK (W)-NH₂ P156 40 C6-HSDAVFTDNYTRLLLKVAAKKYLQSIKNSRTSPPP- NH₂ P157 41 C6-HSDAVFTDQYTRLRKQVAAKKYLQSIKQSRTSPPP- NH₂ P178 42 C6-HSDAVFTDNYTRLRKAAAAKKYLQSIKNSRTSPPP- NH₂ P309 43 C6-HSDAVFTDNYTRLRAibQVAAAibKYLQSIKNSRTS PPP-NH₂

More preferred VPAC2 receptor peptide agonists according to the second aspect of the present invention comprise an amino acid sequence selected from the following;

wherein:

at least one of the Lys residues is covalently attached to a PEG molecule, or

the K(W) is covalently attached to a PEG molecule, or

a combination thereof;

SEQ Agonist ID # NO Peptide P30 29 C6-HSDAVFTDNYTRLRKQVAAKKYLQSIKNSRTSPPP- NH₂ P32 30 Ac-HSDAVFTDNYTRLRKQVAAKKYLQSIKNSRTSPPP- NH₂ P81 32 C6-HSDAVFTDNYTRLRKQVAAKKYLQSIKNSRTSPPPK (E-C16)-NH₂ P95 35 C3-HSDAVFTDNYTRLRKQVAAKKYLQSIKNSRTSPPPK (E-C16)-NH₂ P118 38 C6-HSDAVFTDNYTRLRKQVAAKKYLQSIKNSRTSPPPK (W)-NH₂ P156 40 C6-HSDAVFTDNYTRLLLKVAAKKYLQSIKNSRTSPPP- NH₂ P157 41 C6-HSDAVFTDQYTRLRKQVAAKKYLQSIKQSRTSPPP- NH₂ P309 43 C6-HSDAVFTDNYTRLRAibQVAAAibKYLQSIKNSRTS PPP-NH₂

A more preferred PEGylated VPAC2 receptor peptide agonist according to the second aspect of the invention is:

SEQ Agonist ID # NO Peptide P136 44 C6- HSDAVFTDNYTRLRKQVAAKKYLQSIKNSRTSPPPK (WPEG40K)-NH₂

According to a third aspect of the present invention, there is provided a PEGylated VPAC2 receptor peptide agonist comprising a sequence of the formula:

Formula 13 (SEQ ID NO: 18) Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Thr-Xaa₈-Xaa₉-Xaa₁₀- Thr-Xaa₁₂-Xaa₁₃-Xaa₁₄-Xaa₁₅-Xaa₁₆-Xaa₁₇-Xaa₁₈- Xaa₁₉-Xaa₂₀-Xaa₂₁-Xaa₂₂-Xaa₂₃-Xaa₂₄-Xaa₂₅-Xaa₂₆- Xaa₂₇-Xaa₂₈-Xaa₂₉-Xaa₃₀-Xaa₃₁-Xaa₃₂-Xaa₃₃-Xaa₃₄- Xaa₃₅-Xaa₃₆-Xaa₃₇-Xaa₃₈-Xaa₃₉-Xaa₄₀ wherein:

Xaa₁ is: any naturally occurring amino acid, dH, or is absent;

Xaa₂ is: any naturally occurring amino acid, dA, dS, or Aib;

Xaa₃ is: Asp or Glu;

Xaa₄ is: any naturally occurring amino acid, dA, Aib, or NMeA;

Xaa₅ is: any naturally occurring amino acid, dV, or Aib;

Xaa₆ is: any naturally occurring amino acid;

Xaa₈ is: Asp, Glu, Ala, Lys, Leu, Arg, or Tyr;

Xaa₉ is: Asn, Gln, Asp, Glu, Ser, or Cys;

Xaa₁₀ is: any naturally occurring aromatic amino acid, or Tyr (OMe);

Xaa₁₂ is: hR, Orn, Lys (isopropyl), Aib, Cit, or any naturally occurring amino acid except Pro;

Xaa₁₃ is: Aib, K(CO(CH₂)₂SH), or any naturally occurring amino acid except Pro;

Xaa₁₄ is: hR, Orn, Lys (isopropyl), Aib, Cit, or any naturally occurring amino acid except Pro;

Xaa₁₅ is: hR, Orn, Lys (isopropyl), Aib, K (Ac), Cit, K(W), or any naturally occurring amino acid except Pro;

Xaa₁₆ is: hR, Orn, Lys (isopropyl), Cit, K(CO(CH₂)₂SH), or any naturally occurring amino acid except Pro;

Xaa₁₇ is: Nle, Aib, K(CO(CH₂)₂SH), or any naturally occurring amino acid except Pro;

Xaa₁₈ is: any naturally occurring amino acid;

Xaa₁₉ is: K(CO(CH₂)₂SH), or any naturally occurring amino acid except Pro;

Xaa₂₀ is: hR, Orn, Lys (isopropyl), Aib, K(Ac), Cit, or any naturally occurring amino acid except Pro;

Xaa₂₁ is: hR, Orn, Aib, K(Ac), Cit, or any naturally occurring amino acid except Pro;

Xaa₂₂ is: Aib, Tyr (OMe), or any naturally occurring amino acid except Pro;

Xaa₂₃ is: Aib or any naturally occurring amino acid except Pro;

Xaa₂₄ is: K(CO(CH₂)₂SH), or any naturally occurring amino acid except Pro;

Xaa₂₅ is: Aib, K(CO(CH₂)₂SH), or any naturally occurring amino acid except Pro;

Xaa₂₆ is: K(CO(CH₂)₂SH), or any naturally occurring amino acid except Pro;

Xaa₂₇ is: hR, Lys (isopropyl), Orn, dK, or any naturally occurring amino acid except Pro;

Xaa₂₈ is: any naturally occurring amino acid, Aib, hR, Cit, Orn, dK, or K(CO(CH₂)₂SH);

Xaa₂₉ is: any naturally occurring amino acid, hR, Orn, Cit, Aib, or is absent;

Xaa₃₀ is: any naturally occurring amino acid, hR, Orn, Cit, Aib, or is absent; and

Xaa₃₁ to Xaa₄₀ are any naturally occurring amino acid or are absent;

provided that if Xaa₂₉, Xaa₃₀, Xaa₃₁, Xaa₃₂, Xaa₃₃, Xaa₃₄, Xaa₃₅, Xaa₃₆, Xaa₃₇, Xaa₃₈ or Xaa₃₉ is absent, the next amino acid present downstream is the next amino acid in the peptide agonist sequence and that the peptide agonist comprises at least one amino acid substitution selected from:

Xaa₂ is: dA, Val, Gly, Leu, dS, or Aib;

Xaa₄ is: Ile, Tyr, Phe, Val, Thr, Leu, Trp, dA, Aib, or NMeA;

Xaa₅ is: Leu, Phe, Thr, Trp, Tyr, dV, or Aib;

Xaa₈ is: Leu, Arg, or Tyr;

Xaa₉ is: Glu, Ser, or Cys;

Xaa₁₀ is: Trp;

Xaa₁₂ is: Ala, hR, Aib, Lys (isopropyl), Cit, Gln, or Phe;

Xaa₁₃ is: Phe, Glu, Ala, Aib, Ser, Cys, or K(CO(CH₂)₂SH);

Xaa₁₄ is: Leu, Lys, Ala, hR, Orn, Lys (isopropyl), Phe, Gln, Aib, or Cit;

Xaa₁₅ is: Ala, Arg, Leu, hR, Orn, Lys (isopropyl), Phe, Gln, Aib, K(Ac), Cit, or K(W);

Xaa₁₆ is: Lys, Lys (isopropyl), hR, Orn, Cit, Ser, Cys, or K(CO(CH₂)₂SH);

Xaa₁₇ is: Lys, Aib, Ser, Cys, or K(CO(CH₂)₂SH);

Xaa₁₈ is: Ser, or Cys;

Xaa₁₉ is: K(CO(CH₂)₂SH);

Xaa₂₀ is: Gln, hR, Arg, Ser, Orn, Lys(isopropyl), Ala, Aib, Trp, Thr, Leu, Ile, Phe, Tyr, Val, K(Ac), Cit, or Cys;

Xaa₂₁ is: Arg, Ala, Phe, Aib, Leu, Gln, Orn, hR, K(Ac), Cit, Ser, or Cys;

Xaa₂₂ is: Trp, Thr, Leu, Ile, Val, Tyr(OMe), Ala, Aib, Ser, or Cys;

Xaa₂₃ is: Phe, Ile, Ala, Trp, Thr, Val, Aib, Ser, or Cys;

Xaa₂₄ is: Ser, Cys, or K(CO(CH₂)₂SH);

Xaa₂₅ is: Phe, Ile, Leu, Val, Trp, Gln, Asn, Tyr, Aib, Glu, Cys, or K(CO(CH₂)₂SH);

Xaa₂₆ is: Thr, Trp, Tyr, Phe, Ser, Cys, or K(CO(CH₂)₂SH);

Xaa₂₇ is: hR, Orn, or dK;

Xaa₂₈ is: Pro, Arg, Aib, Orn, hR, Cit, dK, Cys, or K(CO(CH₂)₂SH);

Xaa₂₉ is: hR, Cys, Orn, Cit, or Aib;

Xaa₃₀ is: hR, Cit, Aib, or Orn; and

Xaa₃₁ is: His, or Phe,

and wherein:

at least one of the Cys residues in the peptide agonist is covalently attached to a PEG molecule, or at least one of the Lys residues in the peptide agonist is covalently attached to a PEG molecule, or at least one of the K(CO(CH₂)₂SH) in the peptide agonist is covalently attached to a PEG molecule, or the K(W) in the peptide agonist is covalently attached to a PEG molecule, or the carboxy-terminal amino acid of the peptide agonist is covalently attached to a PEG molecule, or any combination thereof.

Preferably, the PEGylated VPAC2 receptor peptide agonist according to the third aspect of the present invention comprises a sequence of the formula:

Formula 14 (SEQ ID NO: 19) His-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Phe-Thr-Xaa₈-Xaa₉-Xaa₁₀- Thr-Xaa₁₂-Xaa₁₃-Xaa₁₄-Xaa₁₅-Xaa₁₆-Xaa₁₇-Xaa₁₈- Xaa₁₉-Xaa₂₀-Xaa₂₁-Xaa₂₂-Xaa₂₃-Xaa₂₄-Xaa₂₅-Xaa₂₆- Xaa₂₇-Xaa₂₈-Xaa₂₉-Xaa₃₀-Xaa₃₁-Xaa₃₂-Xaa₃₃-Xaa₃₄- Xaa₃₅-Xaa₃₆-Xaa₃₇-Xaa₃₈-Xaa₃₉-Xaa₄₀ wherein:

-   Xaa₂ is: dA, Ser, Val, Gly, Thr, Leu, dS, Pro, or Aib; -   Xaa₃ is: Asp or Glu; -   Xaa₄ is: Ala, Ile, Tyr, Phe, Val, Thr, Leu, Trp, Gly, dA, Aib, or     NMeA; -   Xaa₅ is: Val, Leu, Phe, Ile, Thr, Trp, Tyr, dV, or Aib; -   Xaa₈ is: Asp, Glu, Ala, Lys, Leu, Arg, or Tyr; -   Xaa₉ is: Asn, Gln, Asp, Glu, Ser, or Cys; -   Xaa₁₀ is: Tyr, Trp, or Tyr(OMe); -   Xaa₁₂ is: Arg, Lys, Glu, hR, Orn, Lys (isopropyl), Aib, Cit, Ala,     Leu, Gln, or Phe; -   Xaa₁₃ is: Leu, Phe, Glu, Ala, Aib, Ser, Cys, or K(CO(CH₂)₂SH); -   Xaa₁₄ is: Arg, Leu, Lys, Ala, hR, Orn, Lys (isopropyl), Phe, Gln,     Aib, or Cit; -   Xaa₁₅ is: Lys, Ala, Arg, Glu, Leu, hR, Orn, Lys (isopropyl), Phe,     Gln, Aib, K(Ac), Cit, or K(W); -   Xaa₁₆ is: Gln, Lys, Glu, Ala, hR, Orn, Lys (isopropyl), Cit, Ser,     Cys, or K(CO(CH₂)₂SH); -   Xaa₁₇ is: Val, Ala, Leu, Ile, Met, Nle, Lys, Aib, Ser, Cys, or     K(CO(CH₂)₂SH); -   Xaa₁₈ is: Ala, Ser, or Cys; -   Xaa₁₉ is: Val, Ala, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn,     Gln, Arg, Ser, Thr, Trp, Tyr, Cys, Asp, or K(CO(CH₂)₂SH); -   Xaa₂₀ is: Lys, Gln, hR, Arg, Ser, His, Orn, Lys (isopropyl), Ala,     Aib, Trp, Thr, Leu, Ile, Phe, Tyr, Val, K(Ac), Cit, or Cys; -   Xaa₂₁ is: Lys, His, Arg, Ala, Phe, Aib, Leu, Gln, Orn, hR, K(Ac),     Cit, Ser, or Cys; -   Xaa₂₂ is: Tyr, Trp, Phe, Thr, Leu, Ile, Val, Tyr(OMe), Ala, Aib,     Ser, or Cys; -   Xaa₂₃ is: Leu, Phe, Ile, Ala, Trp, Thr, Val, Aib, Ser, or Cys; -   Xaa₂₄ is: Gln, Glu, Asn, Ser, Cys, or K(CO(CH₂)₂SH); -   Xaa₂₅ is: Ser, Asp, Phe, Ile, Leu, Thr, Val, Trp, Gln, Asn, Tyr,     Aib, Glu, Cys, or K(CO(CH₂)₂SH); -   Xaa₂₆ is: Ile, Leu, Thr, Val, Trp, Tyr, Phe, Ser, Cys, or     K(CO(CH₂)₂SH); -   Xaa₂₇ is: Lys, hR, Arg, Gln, Ala, Asp, Glu, Phe, Gly, His, Ile, Met,     Asn, Ser, Thr, Val, Trp, Tyr, Lys (isopropyl), Cys, Leu, Orn, or dK; -   Xaa₂₈ is: Asn, Asp, Gln, Lys, Arg, Aib, Orn, hR, Cit, Pro, dK, Cys,     or K(CO(CH₂)₂SH); -   Xaa₂₉ is: Lys, Ser, Arg, Asn, hR, Ala, Asp, Glu, Phe, Gly, His, Ile,     Leu, Met, Pro, Gln, Thr, Val, Trp, Tyr, Cys, Orn, Cit, Aib or is     absent; -   Xaa₃₀ is: Arg, Lys, Ile, Ala, Asp, Glu, Phe, Gly, His, Leu, Met,     Asn, Pro, Gln, Ser, Thr, Val, Trp, Tyr, Cys, hR, Cit, Aib, Orn, or     is absent; -   Xaa₃₁ is: Tyr, His, Phe, Thr, Cys, or is absent; -   Xaa₃₂ is: Ser, Cys, or is absent; -   Xaa₃₃ is: Trp or is absent; -   Xaa₃₄ is: Cys or is absent; -   Xaa₃₅ is: Glu or is absent; -   Xaa₃₆ is: Pro or is absent; -   Xaa₃₇ is: Gly or is absent; -   Xaa₃₈ is: Trp or is absent; -   Xaa₃₉ is: Cys or is absent; and -   Xaa₄₀ is: Arg or is absent

provided that if Xaa₂₉, Xaa₃₀, Xaa₃₁, Xaa₃₂, Xaa₃₃, Xaa₃₄, Xaa₃₅, Xaa₃₆, Xaa₃₇, Xaa₃₈, or Xaa₃₉ is absent, the next amino acid present downstream is the next amino acid in the peptide agonist sequence,

and that the peptide agonist comprises at least one amino acid substitution selected from:

-   Xaa₂ is: dA, Val, Gly, Leu, dS, or Aib; -   Xaa₄ is: Ile, Tyr, Phe, Val, Thr, Leu, Trp, dA, Aib, or NMeA; -   Xaa₅ is: Leu, Phe, Thr, Trp, Tyr, dV, or Aib; -   Xaa₈ is: Leu, Arg, or Tyr; -   Xaa₉ is: Glu, Ser, or Cys; -   Xaa₁₀ is: Trp; -   Xaa₁₂ is: Ala, hR, Aib, Lys (isopropyl), Cit, Gln, or Phe; -   Xaa₁₃ is: Phe, Glu, Ala, Aib, Ser, Cys, or K(CO(CH₂)₂SH); -   Xaa₁₄ is: Leu, Lys, Ala, hR, Orn, Lys (isopropyl), Phe, Gln, Aib, or     Cit; -   Xaa₁₅ is: Ala, Arg, Leu, hR, Orn, Lys (isopropyl), Phe, Gln, Aib,     K(Ac), Cit, or K(W); -   Xaa₁₆ is: Lys, Lys (isopropyl), hR, Orn, Cit, Ser, Cys, or     K(CO(CH₂)₂SH); -   Xaa₁₇ is: Lys, Aib, Ser, Cys, or K(CO(CH₂)₂SH); -   Xaa₁₈ is: Ser, or Cys; -   Xaa₁₉ is: K(CO(CH₂)₂SH); -   Xaa₂₀ is: Gln, hR, Arg, Ser, Orn, Lys(isopropyl), Ala, Aib, Trp,     Thr, Leu, Ile, Phe, Tyr, Val, K(Ac), Cit, or Cys; -   Xaa₂₁ is: Arg, Ala, Phe, Aib, Leu, Gln, Orn, hR, K (Ac), Cit, Ser,     or Cys; -   Xaa₂₂ is: Trp, Thr, Leu, Ile, Val, Tyr (OMe), Ala, Aib, Ser, or Cys; -   Xaa₂₃ is: Phe, Ile, Ala, Trp, Thr, Val, Aib, Ser, or Cys; -   Xaa₂₄ is: Ser, Cys, or K(CO(CH₂)₂SH); -   Xaa₂₅ is: Phe, Ile, Leu, Val, Tip, Gln, Asn, Tyr, Aib, Glu, Cys, or     K(CO(CH₂)₂SH); -   Xaa₂₆ is: Thr, Trp, Tyr, Phe, Ser, Cys, or K(CO(CH₂)₂SH); -   Xaa₂₇ is: hR, Orn, or dK; -   Xaa₂₈ is: Pro, Arg, Aib, Orn, hR, Cit, dK, Cys, or K(CO(CH₂)₂SH); -   Xaa₂₉ is: hR, Cys, Orn, Cit, or Aib; -   Xaa₃₀ is: hR, Cit, Aib, or Orn; and -   Xaa₃₁ is: His or Phe,     and wherein:     at least one of the Cys residues in the peptide agonist is     covalently attached to a PEG molecule, or     at least one of the Lys residues in the peptide agonist is     covalently attached to a PEG molecule, or     at least one of the K(CO(CH₂)₂SH) in the peptide agonist is     covalently attached to a PEG molecule, or     the K(W) in the peptide agonist is covalently attached to a PEG     molecule, or the carboxy-terminal amino acid of the peptide agonist     is covalently attached to the PEG molecule, or     any combination thereof.

According to a fourth aspect of the present invention, there is provided a PEGylated VPAC2 receptor peptide agonist of the present invention for use as a medicament.

According to a further aspect of the present invention, there is provided the use of a PEGylated VPAC2 receptor peptide agonist of the present invention for the manufacture of a medicament for the treatment of non-insulin dependent diabetes.

According to a further aspect of the present invention, there is provided the use of a PEGylated VPAC2 receptor peptide agonist of the present invention for the manufacture of a medicament for the treatment of insulin dependent diabetes.

Alternative embodiments of the present invention are described below.

A first alternative embodiment of the present invention is a PEGylated VPAC2 receptor peptide agonist comprising a sequence of the formula:

Formula 4 (SEQ ID NO: 7) Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Thr-Xaa₈-Xaa₉-Xaa₁₀- Thr-Xaa₁₂-Xaa₁₃-Xaa₁₄-Xaa₁₅-Xaa₁₆-Xaa₁₇-Ala-Xaa₁₉- Xaa₂₀-Xaa₂₁-Xaa₂₂-Leu-Xaa₂₄-Xaa₂₅-Xaa₂₆-Xaa₂₇- Xaa₂₈-Xaa₂₉-Xaa₃₀-Xaa₃₁-Xaa₃₂-Xaa₃₃-Xaa₃₄-Xaa₃₅- Xaa₃₆-Xaa₃₇-Xaa₃₈-Xaa₃₉-Xaa₄₀ wherein:

-   Xaa₁ is: His or is absent; -   Xaa₂ is: dA, Ser, Val, Gly, Thr, Leu, dS, or Pro; -   Xaa₃ is: Asp or Glu; -   Xaa₄ is: Ala, Ile, Tyr, Phe, Val, Thr, Leu, Trp, or Gly; -   Xaa₅ is: Val, Leu, Phe, Ile, Thr, Trp, or Tyr; -   Xaa₆ is: Phe, Ile, Leu, Thr, Val, Trp, or Tyr; -   Xaa₈ is: Asp or Glu; -   Xaa₉ is: Asn, Gln, or Asp; -   Xaa₁₀ is: Tyr or Trp; -   Xaa₁₂ is: Arg, Lys, Glu, hR, Orn, or Lys (isopropyl); -   Xaa₁₃ is: Leu, Phe, Glu, or Ala; -   Xaa₁₄ is: Arg, Leu, Lys, Ala, hR, Orn, or Lys (isopropyl); -   Xaa₁₅ is: Lys, Ala, Arg, Glu, Leu, hR, Orn, or Lys (isopropyl); -   Xaa₁₆ is: Gln, Lys, Glu, Ala, hR, Orn, or Lys (isopropyl); -   Xaa₁₇ is: Val, Ala, Leu, Ile, or Met; -   Xaa₁₉ is: Val, Ala, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn,     Pro, Gln, Arg, Ser, Thr, Trp, Tyr, Cys, or Asp; -   Xaa₂₀ is: Lys, Gln, hR, Arg, Ser, His, Orn, or Lys (isopropyl); -   Xaa₂₁ is: Lys, His, or Arg; -   Xaa₂₂ is: Tyr, Trp, Phe, Thr, Leu, Ile, or Val; -   Xaa₂₄ is: Gln, Glu, or Asn; -   Xaa₂₅ is: Ser, Asp, Phe, Ile, Leu, Thr, Val, Trp, Gln, Asn, or Tyr; -   Xaa₂₆ is: Ile, Leu, Thr, Val, Trp, Tyr, or Phe; -   Xaa₂₇ is: Lys, hR, Arg, Gln, Ala, Asp, Glu, Phe, Gly, His, Ile, Met,     Asn, Pro, Ser, Thr, Val, Trp, Tyr, Lys (isopropyl), Cys, or Leu; -   Xaa₂₈ is: Asn, Asp, Gln, Lys, or Arg; -   Xaa₂₉ is: Lys, Ser, Arg, Asn, hR, Gly, Ala, Asp, Glu, Phe, His, Ile,     Leu, Met, Pro, Gln, Thr, Val, Trp, Tyr, Cys, or is absent; -   Xaa₃₀ is: Arg, Lys, Ile, Gly, Ala, Asp, Glu, Phe, His, Leu, Met,     Asn, Pro, Gln, Ser, Thr, Val, Trp, Tyr, Cys, or is absent; -   Xaa₃₁ is: Tyr, His, Phe, Thr, Cys, or is absent; -   Xaa₃₂ is: Ser, Cys, or is absent; -   Xaa₃₃ is: Trp or is absent; -   Xaa₃₄ is: Cys or is absent; -   Xaa₃₅ is: Glu or is absent; -   Xaa₃₆ is: Pro or is absent; -   Xaa₃₇ is: Gly or is absent; -   Xaa₃₈ is: Trp or is absent; -   Xaa₃₉ is: Cys or is absent; and -   Xaa₄₀ is: Arg or is absent

provided that if Xaa₂₉, Xaa₃₀, Xaa₃₁, Xaa₃₂, Xaa₃₃, Xaa₃₄, Xaa₃₅, Xaa₃₆, Xaa₃₇, Xaa₃₈, or Xaa₃₉ is absent, the next amino acid present downstream is the next amino acid in the sequence

and a C-terminal extension wherein the N-terminus of the C-terminal extension is linked to the C-terminus of the sequence and wherein the C-terminal extension comprises an amino acid sequence selected from the group consisting of:

a) Formula 6 (SEQ ID NO: 11) Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉ wherein:

Xaa₁ is: Ser or absent;

Xaa₂ is: Arg, or absent;

Xaa₃ is: Thr or absent;

Xaa₄ is: Ser or absent;

Xaa₅ is: Pro or absent;

Xaa₆ is: Pro or absent;

Xaa₇ is: Pro or absent;

Xaa₈ is: Lys or absent;

Xaa₉ is: K(E-C₁₆) or absent;

provided that if Xaa₁, Xaa₂, Xaa₃, Xaa₄, Xaa₅, Xaa₆, Xaa₇, or Xaa₈ is absent, the next amino acid present downstream is the next amino acid in the C-terminal extension and wherein the C-terminal amino acid may be amidated; and

b) Formula 5 Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇ (SEQ ID NO: 8) wherein:

Xaa₁ is: Ser or absent;

Xaa₂ is: Arg or absent;

Xaa₃ is: Thr or absent;

Xaa₄ is: Ser or absent;

Xaa₅ is: Pro, Ser, Ala, or absent;

Xaa₆ is: Pro, Ser, Ala, or absent; and

Xaa₇ is: Pro, Ser, Ala, or absent;

provided that if Xaa₁, Xaa₂, Xaa₃, Xaa₄, Xaa₅, or Xaa₆ is absent, the next amino acid present downstream is the next amino acid in the C-terminal extension and wherein the C-terminal amino acid may be amidated,

and wherein:

at least one of the Cys residues is covalently attached to a PEG molecule, or

at least one of the Lys residues is covalently attached to a PEG molecule, or

the carboxy-terminal amino acid is covalently attached to a PEG molecule, or

any combination thereof.

Another alternative embodiment of the present invention is a PEGylated VPAC2 receptor peptide agonist comprising a sequence of the formula:

Formula 2 (SEQ ID NO: 5) Xaa₁-Xaa₂-Asp-Xaa₄-Xaa₅-Xaa₆-Thr-Xaa₈-Asn-Xaa₁₀- Thr-Xaa₁₂-Xaa₁₃-Xaa₁₄-Xaa₁₅-Xaa₁₆-Xaa₁₇-Ala-Xaa₁₉- Xaa₂₀-Xaa₂₁-Xaa₂₂-Leu-Xaa₂₄-Xaa₂₅-Xaa₂₆-Xaa₂₇- Xaa₂₈-Xaa₂₉-Xaa₃₀-Xaa₃₁ wherein:

Xaa₁ is: His or is absent;

Xaa₂ is: dA, Ser, Val, Gly, Thr, Leu, dS, or Pro;

Xaa₄ is: Ala, Ile, Tyr, Phe, Val, Thr, Leu, Trp, or Gly;

Xaa₅ is: Val, Leu, Phe, Ile, Thr, Trp, or Tyr;

Xaa₆ is: Phe, Ile, Leu, Thr, Val, Trp, or Tyr;

Xaa₈ is: Asp;

Xaa₁₀ is: Tyr or Trp;

Xaa₁₂ is: Arg or Lys;

Xaa₁₃ is: Leu, Phe, Glu, or Ala;

-   Xaa₁₄is: Arg, Leu, Uds or Ala:

Xaa₁₅ is: Lys, Ala, Arg, Glu, or Leu;

Xaa₁₆ is: Gln, Lys, or Ala;

Xaa₁₇ is: Val, Ala, Leu, or Met;

Xaa₁₉ is: Ala or Leu;

Xaa₂₀ is: Lys, Gln, hR, Arg, or Ser;

Xaa₂₁ is: Lys or Arg;

Xaa₂₂ is: Tyr, Trp, Phe, Thr, Leu, Ile, or Val;

Xaa₂₄ is: Gln or Asn;

Xaa₂₅ is: Ser, Phe, Ile, Leu, Thr, Val, Trp, Gln, Asn, or Tyr;

Xaa₂₆ is: Ile, Leu, Thr, Val, Trp, Tyr, or Phe;

Xaa₂₇ is: Lys, hR, Arg, Gln, or Leu;

Xaa₂₈ is: Asn, Lys, or Arg;

Xaa₂₉ is: Lys, Ser, Arg, Asn, hR, or is absent;

Xaa₃₀ is: Arg, Lys, Ile, or is absent; and

Xaa₃₁ is: Tyr, His, Phe, or is absent,

provided that if Xaa₂₉ is absent then Xaa₃₀ and Xaa₃₁ are also absent and if Xaa₃₀ is absent then Xaa₃₁ is absent,

and a C-terminal extension wherein the N-terminus of the C-terminal extension is linked to the C-terminus of the sequence and wherein the C-terminal extension comprises an amino acid sequence of the Formula 6 (SEQ ID NO: 11), provided that if Xaa₁, Xaa₂, Xaa₃, Xaa₄, Xaa₅, Xaa₆, Xaa₇, or Xaa₈ of Formula 6 (SEQ ID NO: 11) is absent, the next amino acid present downstream is the next amino acid in the C-terminal extension and wherein the C-terminal amino acid may be amidated

and wherein:

at least one of the Cys residues is covalently attached to a PEG molecule, or

at least one of the Lys residues is covalently attached to a PEG molecule, or

the carboxy-terminal amino acid is covalently attached to a PEG molecule, or

any combination thereof.

Yet another alternative embodiment of the present invention is a PEGylated VPAC2 receptor peptide agonist comprising a sequence of the formula:

Formula 3 (SEQ ID NO: 6) His-Xaa₂-Xaa₃-Ala-Val-Phe-Thr-Xaa₈-Xaa₉-Tyr-Thr- Xaa₁₂-Leu-Arg-Xaa₁₅-Xaa₁₆-Xaa₁₇-Ala-Xaa₁₉-Xaa₂₀- Xaa₂₃-Tyr-Leu-Xaa₂₄-Xaa₂₅-Xaa₂₆-Xaa₂₇-Xaa₂₈-Xaa₂₉- Xaa₃₀-Xaa₃₁-Xaa₃₂-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇- Xaa₃₈-Xaa₃₉-Xaa₄₀ wherein:

-   Xaa₂ is: Ser or Thr; -   Xaa₃ is: Asp or Glu; -   Xaa₈ is: Asp or Glu; -   Xaa₉ is: Asn, Gln, or Asp; -   Xaa₁₂ is: Arg, Lys, or Glu; -   Xaa₁₅ is: Lys or Glu; -   Xaa₁₆ is: Gln or Glu; -   Xaa₁₇ is: Met, Leu, Ile, or Val; -   Xaa₁₉ is: Val, Ala, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn,     Pro, Gln, Arg, Ser, Thr, Trp, Tyr, Cys, or Asp; -   Xaa₂₀ is: Lys or His; -   Xaa₂₁ is: Lys or His; -   Xaa₂₄ is: Asn, Gln, or Glu; -   Xaa₂₅ is: Ser, Asp, or Thr; -   Xaa₂₆ is: Ile or Leu; -   Xaa₂₇ is: Leu, Lys, Ala, Asp, Glu, Phe, Gly, His, Ile, Met, Asn,     Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr, or Cys; -   Xaa₂₈ is: Asn, Asp, Gln, or Lys; -   Xaa₂₉ is: Gly, Lys, Ala, Asp, Glu, Phe, His, Ile, Leu, Met, Asn,     Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr, Cys, or is absent; -   Xaa₃₀ is: Gly, Arg, Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met,     Asn, Pro, Gln, Ser, Thr, Val, Trp, Tyr, Cys, or is absent; -   Xaa₃₁ is: Thr, Tyr, Cys, or is absent; -   Xaa₃₂ is: Ser, Cys, or is absent; -   Xaa₃₃ is: Trp or is absent; -   Xaa₃₄ is: Cys or is absent; -   Xaa₃₅ is: Glu or is absent; -   Xaa₃₆ is: Pro or is absent; -   Xaa₃₇ is: Gly or is absent; -   Xaa₃₈ is: Trp or is absent; -   Xaa₃₉ is: Cys or is absent; -   Xaa₄₀ is: Arg or is absent;

provided that if Xaa₂₉, Xaa₃₀, Xaa₃₁, Xaa₃₂, Xaa₃₃, Xaa₃₄, Xaa₃₅, Xaa₃₆, Xaa₃₇, Xaa₃₈, or Xaa₃₉ is absent, the next amino acid present downstream is the next amino acid in the sequence;

and a C-terminal extension wherein the N-terminus of the C-terminal extension is linked to the C-terminus of the sequence and wherein the C-terminal extension comprises an amino acid sequence selected from the group consisting of: a) Formula 6 (SEQ ID NO: 11), provided that if Xaa₁, Xaa₂, Xaa₃, Xaa₄, Xaa₅, Xaa₆, Xaa₇, or Xaa₈ in Formula 6 (SEQ ID NO: 11) is absent, the next amino acid present downstream is the next amino acid in the C-terminal extension and wherein the C-terminal amino acid may be amidated;

and b) Formula 5 (SEQ ID NO: 8), provided that if Xaa₁, Xaa₂, Xaa₃, Xaa₄, Xaa₅, or Xaa₆ is absent in Formula 5 (SEQ ID NO: 8), the next amino acid present downstream is the next amino acid in the C-terminal extension and wherein the C-terminal amino acid may be amidated,

and wherein:

at least one of the Cys residues is covalently attached to a PEG molecule, or

at least one of the Lys residues is covalently attached to a PEG molecule, or

the carboxy-terminal amino acid is covalently attached to a PEG molecule, or

any combination thereof.

Another alternative embodiment of the present invention is a PEGylated VPAC2 receptor peptide agonist comprising a sequence of the formula:

Formula 1 (SEQ ID NO: 4) His-Xaa₂-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr- Xaa₁₂-Leu-Xaa₁₄-Xaa₁₅-Xaa₁₆-Xaa₁₇-Ala-Xaa₁₉-Xaa₂₀- Xaa₂₁-Tyr-Leu-Xaa₂₄-Xaa₂₅-Xaa₂₆-Xaa₂₇-Asn-Xaa₂₉- Xaa₃₀-Xaa₃₁

wherein:

Xaa₂ is: Ser, Val, dA, or dS;

Xaa₁₂ is: Arg, Lys, hR, Orn, or Lys (isopropyl);

Xaa₁₄ is: Arg, Leu, Lys, hR, Orn, or Lys (isopropyl);

Xaa₁₅ is: Lys, Ala, Arg, hR, Orn, or Lys (isopropyl);

Xaa₁₆ is: Gln, Lys, Ala, hR, Orn, or Lys (isopropyl);

Xaa₁₇ is: Met, Val, Ala, or Leu;

Xaa₁₈ is: Val, Ala or Leu;

Xaa₂₀ is: Lys, Gln, Arg, hR, Orn, or Lys (isopropyl);

Xaa₂₁ is: Lys or Arg;

Xaa₂₄ is: Asn or Gln;

Xaa₂₅ is: Ser, Phe, Ile, Leu, Thr, Val, Trp, Gln, Asn, or Tyr;

Xaa₂₆ is: Ile, Leu, Thr, Val, Trp, Tyr, or Phe;

Xaa₂₇ is: Leu, hR, Arg, Lys, or Lys (isopropyl);

Xaa₂₉ is: Lys, Ser, Arg, hR, or absent;

Xaa₃₀ is: Arg, Lys, or absent; and

Xaa₃₁ is: Tyr, Phe, or absent,

provided that at least one Xaa selected from the group consisting of: Xaa₂, Xaa₁₄, Xaa₁₅, Xaa₁₆, Xaa₁₇, Xaa₂₀, Xaa₂₅, Xaa₂₆, Xaa₂₇, and Xaa₃₁ is an amino acid that differs from the amino acid at the corresponding position in SEQ ID NO: 1,

provided that if Xaa₂₉ is absent then Xaa₃₀ and Xaa₃₁ are also absent, and if Xaa₃₀ is absent then Xaa₃₁ is absent

and a C-terminal extension wherein the N-terminus of the C-terminal extension is linked to the C-terminus of the sequence and wherein the C-terminal extension comprises an amino acid sequence of the Formula 5 (SEQ ID NO: 8), provided that if Xaa₁, Xaa₂, Xaa₃, Xaa₄, Xaa₅, or Xaa₆ of Formula 5 (SEQ ID NO: 8) is absent, the next amino acid present downstream is the next amino acid in the C-terminal extension and wherein the C-terminal amino acid may be amidated,

and wherein:

at least one of the Cys residues is covalently attached to a PEG molecule, or

at least one of the Lys residues is covalently attached to a PEG molecule, or

the carboxy-terminal amino acid is covalently attached to a PEG molecule, or

any combination thereof.

A further alternative embodiment of the present invention is a PEGylated VPAC2 receptor peptide agonist comprising a sequence of the formula:

Formula 1 (SEQ ID NO: 4) His-Xaa₂-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr- Xaa₁₂-Leu-Xaa₁₄-Xaa₁₅-Xaa₁₆-Xaa₁₇-Ala-Xaa₁₉-Xaa₂₀- Xaa₂₁-Tyr-Leu-Xaa₂₄-Xaa₂₅-Xaa₂₆-Xaa₂₇-Asn-Xaa₂₉- Xaa₃₀-Xaa₃₁

wherein:

Xaa₂ is: Ser, Val, dA, or dS;

Xaa₁₂ is: Arg, Lys, hR, Orn, or Lys (isopropyl);

Xaa₁₄ is: Arg, Leu, Lys, hR, Orn, or Lys (isopropyl);

Xaa₁₅ is: Lys, Ala, Arg, hR, Orn, or Lys (isopropyl);

Xaa₁₆ is: Gln, Lys, Ala, hR, Orn, or Lys (isopropyl);

Xaa₁₇ is: Met, Val, Ala, or Leu;

Xaa₁₉ is: Val, Ala or Leu;

Xaa₂₀ is: Lys, Gln, Arg, hR, Orn, or Lys (isopropyl);

Xaa₂₁ is: Lys or Arg;

Xaa₂₄ is: Asn or Gln;

Xaa₂₅ is: Ser, Phe, Ile, Leu, Thr, Val, Trp, Gln, Asn, or Tyr;

Xaa₂₆ is: Ile, Leu, Thr, Val, Trp, Tyr, or Phe;

Xaa₂₇ is: Leu, hR, Arg, Lys, or Lys (isopropyl);

Xaa₂₉ is: Lys, Ser, Arg, hR, or absent;

Xaa₃₀ is: Arg, Lys, or absent; and

-   Xaa₃₁ is: Tyr, Phe, or absent,

wherein the sequence has at least one of the following Xaas:

Xaa₂ is: Val or dA;

Xaa₁₄ is: Leu;

Xaa₁₅ is: Ala;

Xaa₁₆ is: Lys;

Xaa₁₇ is: Ala;

Xaa₂₀ is: Gln;

Xaa₂₅ is: Phe, Ile, Leu, Val, Trp, or Tyr;

Xaa₂₆ is: Thr, Trp, or Tyr;

Xaa₂₇ is: hR; and

Xaa₂₈ is: Phe,

and provided that if Xaa₂₉ is absent then Xaa₃₀ and Xaa₃₁ are absent, and if Xaa₃₀ is absent then Xaa₃₁ is absent. This embodiment can further comprise a C-terminal extension wherein the N-terminus of the C-terminal extension is linked to the C-terminus of the peptide comprising Formula 1 (SEQ ID NO:4) and wherein the C-terminal extension comprises an amino acid sequence of the Formula 5 (SEQ ID NO: 8), provided that if Xaa₁, Xaa₂, Xaa₃, Xaa₄, Xaa₅, or Xaa₆ of Formula 5 (SEQ ID NO: 8) is absent, the next amino acid present downstream is the next amino acid in the C-terminal extension and wherein the C-terminal amino acid may be amidated.

Additional alternative embodiments of the present invention include a VPAC2 receptor peptide agonist further comprising a N-terminal modification linked to the N-terminus of the peptide sequence wherein the N-terminal modification involves acylation, alkylation, acetylation, a carbobenzoyl group, a succinimide group, a sulfonamide group, a carbamate group, or a urea group.

Other alternative embodiments of the present invention include a VPAC2 receptor peptide agonist further comprising a N-terminal modification linked to the N-terminus of the peptide sequence wherein the N-terminal modification is selected from the group consisting of D-histidine or isoleucine Alternative embodiments of the present invention also include a VPAC2 receptor peptide agonist further comprising a N-terminal modification linked to the N-terminus of the peptide sequence wherein the N-terminal modification is selected from the group consisting of acetyl, propionyl, butyryl, pentanoyl, hexanoyl, Met, 3-phenylpropionyl, phenylacetyl, benzoyl, or norleucine.

The VPAC2 receptor peptide agonists of the present invention, therefore, have the advantage that they have enhanced selectivity, potency and/or stability over known VPAC2 receptor peptide agonists. In particular, the addition of the C-terminal extension sequence surprisingly increased VPAC2 receptor selectivity as well as increasing proteolytic stability. The covalent attachment of one or more molecules of PEG to particular residues of a VPAC2 receptor peptide agonist results in a biologically active, PEGylated VPAC2 receptor peptide agonist with an extended half-life and reduced clearance when compared to that of non-PEGylated VPAC2 receptor peptide agonists.

A “selective VPAC2 receptor peptide agonist” of the present invention is a peptide that selectively activates the VPAC2 receptor to induce insulin secretion. Preferably, the sequence for a selective VPAC2 receptor peptide agonist of the present invention has from about twenty-eight to about thirty-five naturally occurring and/or non-naturally occurring amino acids and may or may not additionally comprise a C-terminal extension. More preferably, the selective VPAC2 receptor peptide agonist has from twenty-eight to thirty-one naturally occurring and/or non-naturally occurring amino acids and may or may not additionally comprise a C-terminal extension.

A “selective PEGylated VPAC2 receptor peptide agonist” is a selective VPAC2 receptor peptide agonist covalently attached to one or more molecules of polyethylene glycol (PEG), or a derivative thereof, wherein each PEG is attached to a cysteine or lysine amino acid, to a K(W) or K(CO(CH₂)₂SH), or to the carboxy terminus of a peptide.

Selective PEGylated VPAC2 receptor peptide agonists may have a C-terminal extension. The “C-terminal extension” of the present invention comprises a sequence having from one to ten naturally occurring or non-naturally occurring amino acids linked to the C-terminus of the peptide agonist sequence of Formula 7, 9, 10 or 11 at the N-terminus of the C-terminal extension via a peptide bond. Any one of the Cys, Lys, K(W), or K(CO(CH₂)₂SH) residues in the C-terminal extension can be covalently attached to a PEG molecule, or the carboxy-terminal amino acid of the C-terminal extension can be covalently attached to a PEG molecule.

As used herein, the term “linked to” with reference to the term C-terminal extension, includes the addition or attachment of amino acids or chemical groups directly to the C-terminus of the peptide of Formula 7 (SEQ ID NO: 12), Formula 9 (SEQ ID NO: 14), Formula 10 (SEQ ID NO: 15) or Formula 11 (SEQ ID NO: 16).

Optionally, the selective PEGylated VPAC2 receptor peptide agonist may also have an N-terminal modification. The term “N-terminal modification” as used herein includes the addition or attachment of amino acids or chemical groups directly to the N-terminus of a peptide and the formation of chemical groups, which incorporate the nitrogen at the N-terminus of a peptide.

The N-terminal modification may comprise the addition of one or more naturally occurring or non-naturally occurring amino acids to the PEGylated VPAC2 receptor peptide agonist sequence, preferably there are not more then ten amino acids, with one amino acid being more preferred. Naturally occurring amino acids which may be added to the N-terminus include methionine and isoleucine. A modified amino acid added to the N-terminus may be D-histidine. Alternatively, the following amino acids may be added to the N-terminus: SEQ ID NO: 27, Ser-Trp-Cys-Glu-Pro-Gly-Trp-Cys-Arg, wherein the Arg is linked to the N-terminus of the peptide agonist. Preferably, any amino acids added to the N-terminus are linked to the N-terminus by a peptide bond.

The term “linked to” as used herein, with reference to the term N-terminal modification, includes the addition or attachment of amino acids or chemical groups directly to the N-terminus of the PEGylated VPAC2 receptor agonist. The addition of the above N-terminal modifications may be achieved under normal coupling conditions for peptide bond formation.

The N-terminus of the peptide agonist may also be modified by the addition of an alkyl group (R), preferably a C₁-C₁₆ alkyl group, to form (R)NH—.

Alternatively, the N-terminus of the peptide agonist may be modified by the addition of a group of the formula —C(O)R¹ to form an amide of the formula R¹C(O)NH—. The addition of a group of the formula —C(O)R¹ may be achieved by reaction with an organic acid of the formula R¹COOH. Modification of the N-terminus of an amino acid sequence using acylation is demonstrated in the art (e.g. Gozes et al., J. Pharmacol Exp Ther, 273:161-167 (1995)). Addition of a group of the formula —C(O)R¹ may result in the formation of a urea group (see WO 01/23240, WO 2004/006839) or a carbamate group at the N-terminus. Also, the N-terminus may be modified by the addition of pyroglutamic acid or 6-aminohexanoic acid.

The N-terminus of the peptide agonist may be modified by the addition of a group of the formula —SO₂R⁵, to form a sulfonamide group at the N-terminus.

The N-terminus of the peptide agonist may also be modified by reacting with succinic anhydride to form a succinimide group at the N-terminus. The succinimide group incorporates the nitrogen at the N-terminus of the peptide.

The N-terminus may alternatively be modified by the addition of methionine sulfoxide, biotinyl-6-aminohexanoic acid, or —C(═NH)—NH₂. The addition of —C(═NH)—NH₂ is a guanidation modification, where the terminal NH₂ of the N-terminal amino acid becomes —NH—C(═NH)—NH₂.

Most of the sequences of the present invention including the N-terminal modifications and the C-terminal extensions contain the standard single letter or three letter codes for the twenty naturally occurring amino acids. The other codes used are defined as follows:

-   -   Ac=Acetyl     -   C3=propionyl     -   C6=hexanoyl     -   “d” followed by the single letter amino acid code, e.g. dA=D         isoform (non-naturally occurring) of the respective amino acid,         D-alanine, dS=D Serine, dK=D lysine     -   hR=homoarginine     -   _=position not occupied     -   Aib=amino isobutyric acid     -   CH₂=methylene     -   Met(O)=methionine sulfoxide     -   OMe=methoxy     -   Nle=Nor-leucine     -   NMe=methyl attached to the alpha amino group of an amino acid,         e.g. NMeA=N-methyl alanine, NMeA=N-methyl valine     -   Orn=ornithine     -   Cit=citrulline     -   K(Ac)=ε-acetyl lysine     -   M=methionine     -   I=isoleucine     -   K(E-C₁₆)=(ε-(γ-L-glutamyl(N-α-palmitoyl))-lysine     -   K(W)=ε-(L-tryptophyl)-lysine     -   PEG=polyethylene glycol     -   Biotin-Acp=Biotinyl-6-aminohexanoic acid (6-aminocaproic acid)     -   K(CO(CH₂)₂SH)=ε-(3′-mercaptopropionyl)-lysine

The term “VPAC2” is used to refer to and in conjunction with the particular receptor (Lutz, et al., FEBS Lett., 458: 197-203 (1999); Adamou, et al., Biochem. Biophys. Res. Commun., 209; 385-392 (1995)) that the agonists of the present invention activate. This term also is used to refer to and in conjunction with the agonists of the present invention.

VIP naturally occurs as a single sequence having 28 amino acids. However, PACAP exists as either a 38 amino acid peptide (PACAP-38) or as a 27 amino acid peptide (PACAP-27) with an amidated carboxyl (Miyata, et al., Biochem Biophys Res Commun, 170:643-648 (1990)). The sequences for VIP, PACAP-27, and PACAP-38 are as follows:

Seq. Peptide ID # Sequence VIP SEQ ID HSDAVFTDNYTRLRKQMAVKKYLNSILN NO:1 PACAP-27 SEQ ID HSDGIFTDSYSRYRKQMAVKKYLAAVL-NH₂ NO:2 PACAP-38 SEQ ID HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYQRVKN NO:3 K-NH₂

The term “naturally occurring amino acid” as used herein means the twenty amino acids coded for by the human genetic code (i.e. the twenty standard amino acids). These twenty amino acids are: Alanine, Arginine, Asparagine, Aspartic Acid, Cysteine, Glutamine, Glutamic Acid, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine and Valine.

Examples of “non-naturally occurring amino acids” include both synthetic amino acids and those modified by the body. These include D-amino acids, arginine-like amino acids (e.g., homoarginine), and other amino acids having an extra methylene in the side chain (“homo” amino acids), and modified amino acids (e.g norleucine, lysine (isopropyl)—wherein the side chain amine of lysine is modified by an isopropyl group). Also included are amino acids such as ornithine and amino isobutyric acid.

“Selective” as used herein refers to a PEGylated VPAC2 receptor peptide agonist with increased selectivity for the VPAC2 receptor compared to other known receptors. The degree of selectivity is determined by a ratio of VPAC2 receptor binding affinity to VPAC1 receptor binding affinity and by a ratio of VPAC2 receptor binding affinity to PAC1 receptor binding affinity. Preferably, the agonists of the present invention have a selectivity ratio where the affinity for the VPAC2 receptor is at least 50 times greater than for the VPAC1 and/or for PAC1 receptors. More preferably, this affinity is at least 100 times greater for VPAC2 than VPAC1 and/or for PAC1. Even more preferably, the affinity is at least 200 times greater for VPAC2 than for VPAC1 and/or for PAC1. Still more preferably, the affinity is at least 500 times greater for VPAC2 than for VPAC1 and/or for PAC1. Yet more preferably, the affinity is at least 1000 times greater for VPAC2 than for VPAC1 and/or for PAC1. Binding affinity is determined as described below in Example 4.

“Percent (%) sequence identity” as used herein is used to denote sequences which when aligned have similar (identical or conservatively replaced) amino acids in like positions or regions, where identical or conservatively replaced amino acids are those which do not alter the activity or function of the protein as compared to the starting protein. For example, two amino acid sequences with at least 85% identity to each other have at least 85% similar (identical or conservatively replaced residues) in a like position when aligned optimally allowing for up to 3 gaps, with the proviso that in respect of the gaps a total of not more than 15 amino acid residues is affected. Percent sequence identity may be calculated by determining the number of residues that differ between a peptide encompassed by the present invention and a reference peptide such as P5 (SEQ ID NO: 28), taking that number and dividing it by the number of amino acids in the reference peptide (e.g. 35 amino acids for P5), multiplying the result by 100, and subtracting that resulting number from 100. For example, a sequence having 35 amino acids with four amino acids that are different from VIP would have a percent (%) sequence identity of 89% (e.g. 100−((4/35)×100)). For a sequence that is longer than 35 amino acids, the number of residues that differ from the P5 sequence will include the additional amino acids over 35 for purposes of the aforementioned calculation. For example, a sequence having 37 amino acids, with four amino acids different from the 35 amino acids in the P5 sequence and with two additional amino acids at the carboxy terminus which are not present in the P5 sequence, would have a total of six amino acids that differ from P5. Thus, this sequence would have a percent (%) sequence identity of 83% (e.g. 100−((6/35)×100)). The degree of sequence identity may be determined using methods well known in the art (see, for example, Wilbur, W. J. and Zipman, D. R., Proc. Natl. Acad. Sci. USA 80:726-730 (1983) and Myers E. and Miller W., Comput. Appl. Biosci. 4:11-17 (1988)). One program which may be used in determining the degree of similarity is the MegAlign Lipman-Pearson one pair method (using default parameters) which can be obtained from DNAstar Inc, 1128, Selfpark Street, Madison, Wis., 53715, USA as part of the Lasergene system. Another program, which may be used, is Clustal W. This is a multiple sequence alignment package developed by Thompson et al (Nucleic Acids Research, 22(22):4673-4680 (1994)) for DNA or protein sequences. This tool is useful for performing cross-species comparisons of related sequences and viewing sequence conservation. Clustal W is a general purpose multiple sequence alignment program for DNA or proteins. It produces biologically meaningful multiple sequence alignments of divergent sequences. It calculates the best match for the selected sequences, and lines them up so that the identities, similarities and differences can be seen. Evolutionary relationships can be seen via viewing Cladograms or Phylograms.

The sequence for a selective PEGylated VPAC2 receptor peptide agonist of the present invention is selective for the VPAC2 receptor and preferably has a sequence identity in the range of 60% to 70%, 60% to 65%, 65% to 70%, 70% to 80%, 70% to 75%, 75% to 80%, 80% to 90%, 80% to 85%, 85% to 90%, 90% to 97%, 90% to 95%, or 95% to 97%, with P5 (SEQ ID NO. 28). Preferably, the sequence has a sequence identity of greater than 68% with P5 (SEQ ID NO: 28). More preferably, the sequence has greater than 80% sequence identity with P5 (SEQ ID NO: 28). Even more preferably, the sequence has greater than 85% sequence identity with P5 (SEQ ID NO: 28). Yet more preferably, the sequence has greater than 91% sequence identity with P5 (SEQ ID NO: 28).

The term “C₁-C₁₆ alkyl” as used herein means a monovalent saturated straight, branched or cyclic chain hydrocarbon radical having from 1 to 16 carbon atoms. Thus the term “C₁-C₁₆ alkyl” includes, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-heptyl, n-octyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. The C₁-C₁₆ alkyl group may be optionally substituted with one or more substituents.

The term “C₁-C₆ alkyl” as used herein means a monovalent saturated straight, branched or cyclic chain hydrocarbon radical having from 1 to 6 carbon atoms. Thus the term “C₁-C₆ alkyl” includes, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. The C₁-C₆ alkyl group may be optionally substituted with one or more substituents.

The term “C₂-C₆ alkenyl” as used herein means a monovalent straight, branched or cyclic chain hydrocarbon radical having at least one double bond and having from 2 to 6 carbon atoms. Thus the term “C₂-C₆ alkenyl” includes vinyl, prop-2-enyl, but-3-enyl, pent-4-enyl and isopropenyl. The C₂-C₆ alkenyl group may be optionally substituted with one or more substituents.

The term “C₂-C₆ alkynyl” as used herein means a monovalent straight or branched chain hydrocarbon radical having at least one triple bond and having from 2 to 6 carbon atoms. Thus the term “C₂-C₆ alkynyl” includes prop-2-ynyl, but-3-ynyl and pent-4-ynyl. The C₂-C₆ alkynyl may be optionally substituted with one or more substituents.

The term “halo” or “halogen” means fluorine, chlorine, bromine or iodine.

The term “aryl” when used alone or as part of a group is a 5 to 10 membered aromatic or heteroaromatic group including a phenyl group, a 5 or 6-membered monocyclic heteroaromatic group, each member of which may be optionally substituted with 1, 2, 3, 4 or 5 substituents (depending upon the number of available substitution positions), a naphthyl group or an 8-, 9- or 10-membered bicyclic heteroaromatic group, each member of which may be optionally substituted with 1, 2, 3, 4, 5 or 6 substituents (depending on the number of available substitution positions). Within this definition of aryl, suitable substitutions include C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, amino, hydroxy, halogen, —SH and CF₃.

The term “aryl C₁-C₄ alkyl” as used herein means a C₁-C₄ alkyl group substituted with an aryl. Thus the term “aryl C₁-C₄ alkyl” includes benzyl, 1-phenylethyl (α-methylbenzyl), 2-phenylethyl, 1-naphthalenemethyl or 2-naphthalenemethyl.

The term “naphthyl” includes 1-naphthyl, and 2-naphthyl. 1-naphthyl is preferred.

The term “benzyl” as used herein means a monovalent unsubstituted phenyl radical linked to the point of substitution by a —CH₂— group.

The term “5- or 6-membered monocyclic heteroaromatic group” as used herein means a monocyclic aromatic group with a total of 5 or 6 atoms in the ring wherein from 1 to 4 of those atoms are each independently selected from N, O and S. Preferred groups have 1 or 2 atoms in the ring which are each independently selected from N, O and S. Examples of 5-membered monocyclic heteroaromatic groups include pyrrolyl (also called azolyl), furanyl, thienyl, pyrazolyl (also called 1H-pyrazolyl and 1,2-diazolyl), imidazolyl, oxazolyl (also called 1,3-oxazolyl), isoxazolyl (also called 1,2-oxazolyl), thiazolyl (also called 1,3-thiazolyl), isothiazolyl (also called 1,2-thiazolyl), triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, oxatriazolyl and thiatriazolyl. Examples of 6-membered monocyclic heteroaromatic groups include pyridinyl, pyrimidyl, pyrazinyl, pyridazinyl and triazinyl.

The term “8-, 9- or 10-membered bicyclic heteroaromatic group” as used herein means a fused bicyclic aromatic group with a total of 8, 9 or 10 atoms in the ring system wherein from 1 to 4 of those atoms are each independently selected from N, O and S. Preferred groups have from 1 to 3 atoms in the ring system which are each independently selected from N, O and S. Suitable 8-membered bicyclic heteroaromatic groups include imidazo[2,1-b][1,3]thiazolyl, thieno[3,2-b]thienyl, thieno[2,3-d][1,3]thiazolyl and thieno[2,3-d]imidazolyl. Suitable 9-membered bicyclic heteroaromatic groups include indolyl, isoindolyl, benzofuranyl (also called benzo[b]furanyl), isobenzofuranyl (also called benzo[c]furanyl), benzothienyl (also called benzo[b]thienyl), isobenzothienyl (also called benzo[c]thienyl), indazolyl, benzimidazolyl, 1,3-benzoxazolyl, 1,2-benzisoxazolyl, 2,1-benzisoxazolyl, 1,3-benzothiazolyl, 1,2-benzoisothiazolyl, 2,1-benzoisothiazolyl, benzotriazolyl, 1,2,3-benzoxadiazolyl, 2,1,3-benzoxadiazolyl, 1,2,3-benzothiadiazolyl, 2,1,3-benzothiadiazolyl, thienopyridinyl, purinyl and imidazo[1,2-a]pyridine. Suitable 10-membered bicyclic heteroaromatic groups include quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, quinoxalinyl, 1,5-naphthyridyl, 1,6-naphthyridyl, 1,7-naphthyridyl and 1,8-naphthyridyl.

The term “C₁-C₆ alkoxy” as used herein means a monovalent unsubstituted saturated straight-chain or branched-chain hydrocarbon radical having from 1 to 6 carbon atoms linked to the point of substitution by a divalent O radical. Thus the term “C₁-C₆ alkoxy” includes, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy and tert-butoxy. The C₁-C₆ alkoxy may be optionally substituted with one or more substituents.

The term “PEG” as used herein means a polyethylene glycol molecule. In its typical form. PEG is a linear polymer with terminal hydroxyl groups and has the formula HO—CH₂CH₂—(CH₂CH₂O)n-CH₂CH₂—OH, where n is from about 8 to about 4000. The terminal hydrogen may be substituted with a protective group such as an alkyl or alkanol group. Preferably, PEG has at least one hydroxy group, more preferably it is a terminal hydroxy group. It is this hydroxy group which is preferably activated to react with the peptide. There are many forms of PEG useful for the present invention. Numerous derivatives of PEG exist in the art and are suitable for use in the invention. (See, e.g., U.S. Pat. Nos. 5,445,090; 5,900,461; 5,932,462; 6,436,386; 6,448,369; 6,437,025; 6,448,369; 6,495,659; 6,515,100 and 6,514,491 and Zalipsky, S. Bioconjugate Chem. 6:150-165, 1995). The PEG molecule covalently attached to VPAC2 receptor peptide agonists in the present invention is not intended to be limited to a particular type. The molecular weight of the PEG molecule is preferably from 500-100,000 daltons and more preferably 10,000, 20,000, 30,000, 40,000, 50,000, or 60,000 daltons and most preferably 20,000, or 40,000 daltons. PEG may be linear or branched and PEGylated VPAC2 receptor peptide agonists of the invention may have one, two or three PEG molecules attached to the peptide. It is more preferable that there be one or two PEG molecules per PEGylated VPAC2 receptor peptide agonist, however, when there is more than one PEG molecule per peptide molecule, it is preferred that there be no more than three. It is further contemplated that both ends of the PEG molecule may be homo- or hetero-functionalized for crosslinking two or more VPAC2 receptor peptide agonists together. Where there are two PEG molecules present, the PEG molecules will preferably be 20,000 dalton PEG molecules. However, PEG molecules having a different molecular weight may be used, for example, one 10,000 dalton PEG molecule and one 30,000 PEG molecule.

In the present invention, a PEG molecule may be covalently attached to a Cys or Lys residue or to the C-terminal residue. The PEG molecule may also be covalently attached to a Trp residue which is coupled to the side chain of a Lys residue (K(W)). Alternatively, a K(CO(CH₂)₂SH) group may be PEGylated to form K(CO(CH₂)₂S-PEG). Any Lys residue in the peptide agonist may be substituted for a K(W) or K(CO(CH₂)₂SH), which may then be PEGylated. In addition, any Cys residue in the peptide agonist may be substituted for a modified cysteine residue, for example, hC. The modified Cys residue may be covalently attached to a PEG molecule.

The term “PEGylation” as used herein means the covalent attachment of one or more PEG molecules as described above to the VPAC2 receptor peptide agonists of the present invention.

“Insulinotropic activity” refers to the ability to stimulate insulin secretion in response to elevated glucose levels, thereby causing glucose uptake by cells and decreased plasma glucose levels. Insulinotropic activity can be assessed by methods known in the art, including using experiments that measure VPAC2 receptor binding activity or receptor activation (e.g. insulin secretion by insulinoma cell lines or islets, intravenous glucose tolerance test (IVGTT), intraperitoneal glucose tolerance test (IPGTT), and oral glucose tolerance test (OGTT)). Insulinotropic activity is routinely measured in humans by measuring insulin levels or C-peptide levels. Selective PEGylated VPAC2 receptor peptide agonists of the present invention can have insulinotropic activity.

“In vitro potency” as used herein is the measure of the ability of a peptide to activate the VPAC2 receptor in a cell-based assay. In vitro potency is expressed as the “EC₅₀” which is the effective concentration of compound that results in a 50% of maximum increase in activity in a single dose-response experiment. For the purposes of the present invention, in vitro potency is determined using two different assays: DiscoveRx and Alpha Screen. See Example 3 for further details of these assays. While these assays are performed in different ways, the results demonstrate a general correlation between the two assays.

The term “plasma half-life” refers to the time in which half of the relevant molecules circulate in the plasma prior to being cleared. An alternatively used term is “elimination half-life.” The term “extended” or “longer” used in the context of plasma half-life or elimination half-life indicates there is a statistically significant increase in the half-life of a PEGylated VPAC2 receptor peptide agonist relative to that of the reference molecule (e.g., the non-PEGylated form of the peptide or the native peptide) as determined under comparable conditions. Preferably a PEGylated VPAC2 receptor peptide agonist of the present invention has an elimination half-life of at least one hour, more preferably at least 3, 5, 7, 10, 15, 20 or 24 hours and most preferably at least 48 hours. The half-life reported herein is the elimination half-life; it is that which corresponds to the terminal log-linear rate of elimination. The person skilled in the art appreciates that half-life is a derived parameter that changes as a function of both clearance and volume of distribution.

Clearance is the measure of the body's ability to eliminate a drug. As clearance decreases due, for example, to modifications to a drug, half-life would be expected to increase. However, this reciprocal relationship is exact only when there is no change in the volume of distribution. A useful approximate relationship between the terminal log-linear half-life (t_(1/2)), clearance (C), and volume of distribution (V) is given by the equation: t_(1/2)≈0.693 (V/C). Clearance does not indicate how much drug is being removed but, rather, the volume of biological fluid such as blood or plasma that would have to be completely freed of drug to account for the elimination. Clearance is expressed as a volume per unit of time. The PEGylated VPAC2 receptor peptide agonists of the present invention preferably have a clearance value of 200 ml/h/kg or less, more preferably 180, 150, 120, 100, 80, 60 ml/h/kg or less and most preferably 50, 40 or 20 ml/h/kg or less.

According to a preferred embodiment of the present invention, there is provided a PEGylated VPAC2 receptor peptide agonist comprising an amino acid sequence of Formula 7 (SEQ ID NO. 12), Formula 9 (SEQ ID NO 14) or Formula 10 (SEQ ID NO 15) wherein Xaa₃ is Asp or Glu, Xaa₈ is Asp or Glu, Xaa₈ is Asn or Gln, Xaa₁₂ is Arg, hR, Lys, or Orn, Xaa₁₄ is Arg, Gln, Aib, hR, Orn, Cit, Lys, Ala, or Leu, Xaa₁₅ is Lys, Leu, Aib, Orn or Arg, Xaa₁₆ is Gln, or Lys, Xaa₁₇ is Val, Leu, Ala, Ile, Lys, or Nle, Xaa₂₀ is Lys, Val, Leu, Aib, Ala, Gln or Arg, Xaa₂₁ is Lys, Aib, Orn, Ala, Gln or Arg, Xaa₂₇ is Lys, Orn, hR or Arg, and Xaa₂₈ is Asn, Gln, Lys, hR, Aib, Pro, or Orn, and a C-terminal extension comprising an amino acid sequence of (SEQ ID NO: 13), more preferably the C-terminal extension comprises an amino acid sequence of Formula 12 (SEQ ID NO:17).

According to yet another preferred embodiment of the present invention, the PEGylated VPAC2 receptor peptide agonist comprises a sequence of the Formula 7 (SEQ ID NO. 12), Formula 9 (SEQ ID NO 14) or Formula 10 (SEQ ID NO 15) wherein Xaa₉ is Gln, or Asn, Xaa₁₄ is Arg, or Leu, Xaa₁₅ is Lys, Leu, or Aib, Xaa₁₆ is Gln, Lys, or Ala, Xaa₁₇ is Val, or Ala, Xaa₂₀ is Lys, or Aib, and Xaa₂₈ is Asn, or Gln, and further comprises a C-terminal extension of Formula 12 (SEQ ID NO: 17).

It is more preferred that the C-terminal extension is selected from: SRTSPPP (SEQ ID NO: 9), SRTSPPP-NH₂ (SEQ ID NO: 10), SRTSPPPK(W) (SEQ ID NO: 23) and SRTSPPPK(W)-NH₂ (SEQ ID NO: 24).

According to another embodiment of the present invention, the PEGylated VPAC2 receptor peptide agonist comprises a sequence of the Formula 7 (SEQ ID NO. 12), Formula 9 (SEQ ID NO 14) or Formula 10 (SEQ ID NO 15) wherein Xaa₁₄ is Leu, Xaa₁₅ is Ala, Xaa₁₆ is Lys, Xaa₁₇ is Leu and Xaa₂₀ is Gln.

According to another preferred embodiment of the present invention, there is provided a PEGylated VPAC2 receptor peptide agonist comprising an amino acid sequence of Formula 7 (SEQ ID NO. 12) or Formula 9 (SEQ ID NO 14) wherein Xaa₃₀ and Xaa₃₁ are absent, and a C-terminal extension comprising an amino acid sequence of Formula 12 (SEQ ID NO: 17).

Alternatively, in yet another preferred embodiment of the present invention, the PEGylated VPAC2 receptor peptide agonist comprises an amino acid sequence of Formula 7 (SEQ ID NO. 12) or Formula 9 (SEQ ID NO 14) wherein Xaa₂₉, Xaa₃₀ and Xaa₃₁ are absent, and a C-terminal extension comprising an amino acid sequence of Formula 12 (SEQ ID NO: 17).

It is more preferred that the C-terminal extension is selected from: SRTSPPP (SEQ ID NO: 9), SRTSPPP-NH₂ (SEQ ID NO: 10), SRTSPPPK(W) (SEQ ID NO: 23) and SRTSPPPK(W)-NH₂ (SEQ ID NO: 24).

According to another preferred embodiment of the present invention, there is provided a PEGylated VPAC2 receptor peptide agonist comprising an amino acid sequence of Formula 7 (SEQ ID NO: 12), Formula 9 (SEQ ID NO: 14) or Formula 10 (SEQ ID NO: 15) wherein either Xaa₁₄ or Xaa₁₅ is Aib and either Xaa₂₀ or Xaa₂₁ is Aib, more preferably Xaa₁₅ is Aib and Xaa₂₀ is Aib, and a C-terminal extension comprising an amino acid sequence of Formula 12 (SEQ ID NO: 17).

According to yet another preferred embodiment of the present invention, there is provided a PEGylated VPAC2 receptor peptide agonist comprising an amino acid sequence of Formula 7 (SEQ ID NO: 12), Formula 9 (SEQ ID NO: 14) or Formula 10 (SEQ ID NO: 15) wherein Xaa₁₅ is Aib, Xaa₂₀ is Aib, and Xaa₁₂, Xaa₂₁, Xaa₂₇, and Xaa₂₈ are all Orn, and a C-terminal extension comprising an amino acid sequence of Formula 12 (SEQ ID NO: 17). In this embodiment, it is especially preferred that Xaa₈ is Glu, Xaa₉ is Gln, and Xaa₁₀ is Tyr(OMe).

According to another preferred embodiment of the present invention, there is provided a PEGylated VPAC2 receptor peptide agonist comprising an amino acid sequence of the Formula 11 (SEQ ID NO: 16) and a C-terminal extension comprising an amino acid sequence of Formula 12 (SEQ ID NO: 17).

In the above preferred embodiments of the present invention, it is especially preferred that the PEGylated VPAC2 receptor peptide agonist further comprises a N-terminal modification, wherein the N-terminal modification is the addition of a group selected from: acetyl, propionyl, butyryl, pentanoyl, hexanoyl, methionine, methionine sulfoxide, 3-phenylpropionyl, phenylacetyl, benzoyl, norleucine, D-histidine, isoleucine, 3-mercaptopropionyl, biotinyl-6-aminohexanoic acid and —C(═NH)NH₂, and more preferably is the addition of acetyl, hexanoyl, propionyl, 3-phenylpropionyl and benzoyl.

In a preferred embodiment, there is provided a PEGylated VPAC2 receptor peptide agonist comprising an amino acid sequence of Formula 7 (SEQ ID NO. 12), Formula 9 (SEQ ID NO 14) or Formula 10 (SEQ ID NO 15) wherein either Xaa₁₄ or Xaa₁₅ is Aib and either Xaa₂₀ or Xaa₂₁ is Aib, more preferably Xaa₁₅ is Aib and Xaa₂₀ is Aib, and a C-terminal extension selected from: SRTSPPP (SEQ ID NO: 9), SRTSPPP-NH₂ (SEQ ID NO: 10), SRTSPPPK(W) (SEQ ID NO: 23) and SRTSPPPK(W)-NH₂ (SEQ ID NO: 24) and wherein the PEGylated VPAC2 receptor peptide agonist further comprises a N-terminal modification which modification is the addition of hexanoyl or acetyl.

In another preferred embodiment, there is provided a PEGylated VPAC2 receptor peptide agonist comprising an amino acid sequence of Formula 7 (SEQ ID NO. 12), Formula 9 (SEQ ID NO 14) or Formula 10 (SEQ ID NO 15) wherein Xaa₁₅ is Aib, Xaa₂₀ is Aib, Xaa₁₂, Xaa₂₁, Xaa₂₇, and Xaa₂₈ are all Orn, Xaa₈ is Glu, Xaa₉ is Gln, and Xaa₁₀ is Tyr(OMe), and a C-terminal extension selected from: SRTSPPP (SEQ ID NO: 9), SRTSPPP-NH₂ (SEQ ID NO: 10), SRTSPPPK(W) (SEQ ID NO: 23) and SRTSPPPK(W)-NH₂ (SEQ ID NO: 24) and wherein the PEGylated VPAC2 receptor peptide agonist further comprises a N-terminal modification which modification is the addition of hexanoyl or acetyl.

In combination with any one of the preferred embodiments described above, it is preferred that there is at least one PEG molecule covalently attached to Xaa₂₅ or any subsequent residue in Formula 7, 9, 10 or 11, and/or there is at least one PEG molecule covalently attached to a residue in the C-terminal extension of the peptide agonist. It is also preferred that one or two of the Cys residues in the peptide agonist are covalently attached to a PEG molecule, or one or two of the Lys residues in the peptide agonist are covalently attached to a PEG molecule.

A preferred alternative sequence for selective PEGylated VPAC2 receptor peptide agonists of the present invention comprises an amino acid sequence of the Formula 1 (SEQ ID NO: 4), provided that if Xaa₂₉ or Xaa₃₀ is absent each amino acid downstream is absent and wherein the C-terminal amino acid may be amidated.

Throughout this specification, with respect to when an Xaa is absent, the next amino acid present downstream is the next amino acid in the sequence or is also absent. For example, if Xaa₂₉ is Lys and Xaa₃₀ is absent, the next amino acid bonded to Lys at position 29 is an amino acid listed for position 31 or absent, and so forth.

Preferably, an alternative selective PEGylated VPAC2 receptor peptide agonist of the present invention has the amino acid sequence of Formula 1 (SEQ ID NO: 4) modified so that from one, two, three, four, five, six, seven, eight, nine, or ten amino acids differ from the amino acid in the corresponding position of SEQ ID NO: 1.

Another alternative preferred sequence for selective PEGylated VPAC2 receptor peptide agonists of the present invention comprises an amino acid sequence of Formula 1 (SEQ ID NO: 4), wherein: Xaa₂ is: Ser, Val, or dA; Xaa₁₂ is: Arg or Lys; Xaa₁₄ is: Arg, Leu, or Lys; Xaa₁₅ is: Lys, Ala, or Arg; Xaa₁₆ is: Gln, Lys, or Ala; Xaa₁₇ is: Met, Val, Ala, or Leu; Xaa₁₉ is: Val, Ala or Leu; Xaa₂₀ is: Lys, Gln, or Arg; Xaa₂₁ is: Lys or Arg; Xaa₂₄ is: Asn or Gln; Xaa₂₅ is: Ser, Phe, Ile, Leu, Thr, Val, Trp, Gln, Asn, or Tyr; Xaa₂₆ is: Ile, Leu, Thr, Val, Trp, or Tyr; Xaa₂₇ is: Leu, hR, Arg, or Lys; Xaa₂₉ is: Lys, Ser, Arg, or absent; Xaa₃₀ is: Arg, Lys, or absent; and Xaa₃₁ is: Tyr, Phe, or absent.

Another alternative preferred sequence for selective PEGylated VPAC2 receptor peptide agonists of the present invention comprises an amino acid sequence of Formula 1 (SEQ ID NO: 4), wherein: Xaa₂ is: Val or dA; Xaa₁₄ is: Leu; Xaa₁₅ is: Ala; Xaa₁₆ is: Lys; Xaa₁₇ is: Ala; Xaa₂₀ is: Gln; Xaa₂₅ is: Phe, Be, Leu, Val, Trp, or Tyr; Xaa₂₆ is: Thr, Trp, or Tyr; Xaa₂₇ is: hR; and Xaa₃₁ is: Phe.

Another alternative preferred sequence for selective PEGylated VPAC2 receptor peptide agonists of the present invention comprises an amino acid sequence of Formula 1 (SEQ ID NO: 4), wherein: Xaa₂ is: Ser, Val, or dA; Xaa₁₂ is: Arg, Lys, hR, Orn, or Lys (isopropyl); Xaa₁₄ is: Arg, Leu, or Lys; Xaa₁₅ is: Lys, Ala, or Arg; Xaa₁₆ is: Gln, Lys, or Ala; Xaa₁₇ is: Met, Val, Ala, or Leu; Xaa₁₉ is: Val, Ala, or Leu; Xaa₂₀ is: Lys, Gln, or Arg; Xaa₂₁ is: Lys or Arg; Xaa₂₄ is: Asn or Gln, Xaa₂₅ is: Ser, Phe, Ile, Leu, Val, Trp, Tyr, Thr, Gln, or Asn; Xaa₂₆ is: Ile, Thr, Trp, Tyr, Leu, or Val; Xaa₂₇ is: Leu, Lys, hR, or Arg; Xaa₂₉ is: Lys, Ser, Arg, hR, or absent; Xaa₃₀ is: Arg, Lys, or absent; and Xaa₃₁ is: Tyr, Phe, or absent.

Yet another alternative preferred sequence for selective PEGylated VPAC2 receptor peptide agonists of the present invention comprises an amino acid sequence of Formula 1 (SEQ ID NO: 4), wherein: Xaa₁₄ is Leu when Xaa₁₅ is Ala and Xaa₁₆ is Lys. Even more preferably, Xaa₁₄ is Leu when Xaa₁₅ is Ala, Xaa₁₆ is Lys, Xaa₁₇ is Leu, and Xaa₂₀ is Gln.

Another alternative preferred sequence for selective PEGylated VPAC2 receptor peptide agonists of the present invention comprises an amino acid sequence of the Formula 2 (SEQ ID NO: 5), provided that if Xaa₂₉ or Xaa₃₀ of Formula 2 (SEQ ID NO: 5) is absent each amino acid downstream is absent and wherein the C-terminal amino acid may be amidated.

Preferably, an alternative selective PEGylated VPAC2 receptor peptide agonist of the present invention has the amino acid sequence of Formula 2 (SEQ ID NO: 5), modified so that from one, two, three, four, five, six, seven, eight, nine, or ten amino acids differ from the amino acid in the corresponding position of SEQ ID NO: 1.

Another alternative preferred sequence for selective PEGylated VPAC2 receptor peptide agonists of the present invention comprises an amino acid sequence of the Formula 3 (SEQ ID NO: 6), provided that if Xaa₂₉, Xaa₃₀, Xaa₃₁, Xaa₃₂, Xaa₃₃, Xaa₃₄, Xaa₃₅, Xaa₃₆, Xaa₃₇, Xaa₃₈, or Xaa₃₉ of Formula 3 (SEQ ID NO: 6) is absent, the next amino acid present downstream is the next amino acid in the peptide sequence and wherein the C-terminal amino acid may be amidated. For example, if Xaa₂₉ is Gly and Xaa₃₀ is absent, the next amino acid bonded to Gly at position 29 is an amino acid listed for position 31 or, if position 31 is also absent, an amino acid listed for position 32 is bonded to Gly at position 29, and so forth. Additionally, for example, if Xaa₂₉ is Gly and Xaa₃₀ through Xaa₄₀ are absent, Gly may be the C-terminal amino acid and may be amidated.

Preferably, an alternative selective PEGylated VPAC2 receptor peptide agonist of the present invention has the amino acid sequence of Formula 3 (SEQ ID NO: 6), modified so that from one, two, three, four, five, six, seven, eight, nine, or ten amino acids differ from the amino acid in the corresponding position of SEQ ID NO: 1.

Preferable alternative sequences for selective PEGylated VPAC2 receptor peptide agonists include:

SEQ ID NO: Peptide 45. HSDAVFTDNYTRLRKQMAVKKYLNSIKK-NH₂ 46. HSDAVFTDNYTRLRKQMAVKKYLNSIKKGGT 47. HSDAVFTENYTKLRKQLAAKKYLNDLLNGGT 48. HSDAVFTDNYTKLRKQLAAKKYLNDILNGGT 49. HSDAVFTENYTKLRKQLAAKKYLNDLKKGGTSWCEPGWCR 50. HSDAVFTDNYTRLRKQLAAKKYLNSIKKGGT 51. HSDAVFTDNYTRLRKQLAAKKYLNDIKNGGT 52. HSDAVFTDNYTRLRKQLAVKKYLNSIKKGGT 53. HSDAVFTDNYTRLRKQMAAKKYLNSIKKGGT 54. HSDAVFTDNYTRLRKQLAVKKYLNDIKNGGT 55. HSDAVFTDNYTRLRKQLAAKKYLNSIKNGGT 56. HSDAVFTDNYTRLRKQLAAKKYLNDIKKKRY 57. HSDAVFTDNYTRLRKQMAVKKYLNSIKK 58. HSDAVFTDNYTRLRKQMAVKKYLNSIKN 59. HSDAVFTDNYTRLRKQMAVKKYLNSILK 60. HSDAVFTDNYTELRKQMAVKKYLNSILN 61. HSDAVFTDNYTRLRKQMAVKKYLNDILN 62. HSDAVFTDNYTRLRKQMAAKKYLNSIKN 63. HSDAVFTDNYTRLRKQMAAKKYLNSILK 64. HSDAVFTDNYTRLRKQMAAKKYLNSIKK 65. HSDAVFTDNYTRLRKQMAAKKYLNSIKKKRY 66. HSDAVFTDNYTRLRKQMAAKKYLNSIKKKR 67. HSDAVFTDNYTRLRKQMAAKKYLNSIKKK 68. HSDAVFTDNYTRLRKQMAAKKYLNSIKNKRY 69. HSDAVFTDNYTRLRKQMAVKKYLNSIKKKRY 70. HSDAVFTDNYTRLRKQMAVKKYLNSIKKKR 71. HSDAVFTDNYTRLRKQMAVKKYLNSIKKK 72. HSDAVFTDNYTRLRKQMAVKKYLNSIKNKRY 73. HSDAVFTDNYTRLRKQVAAKKYLQSIKK 74. HSDAVFTDNYTRLRKQIAAKKYLQTIKK 75. HSDAVFTENYTRLRKQMAVKKYLNSLKK-NH₂ 76. HSDAVFTDNYTRLRKQLAAKKYLNDILKGGT 77. HSDAVFTDNYTRLRKQLAAKKYLNDILNGGT 78. HSDAVFTDNYTRLRKQLAVKKYLNDILKGGT 79. HSDAVFTDNYTRLRKQVAAKKYLNSIKK 80. HSDAVFTDNYTRLRKQMAAKKYLNSIKNKR 81. HSDAVFTDNYTRLRKQVAAKKYLQSIKNKRY 82. HSDAVFTDNYTRLRKQLAAKKYLNTIKNKRY 83. HSDAVFTDNYTRLRKQVAAKKYLNSIKNKRY 84. HSDAVFTDNYTRLRKQMAAKKYLQSIKNKRY 85. HSDAVFTDNYTRLRKQMAAKKYLNTIKNKRY 86. HSDAVFTDQYTRLRKQMAAKKYLNSIKNKRY 87. HSDAVFTDQYTRLRKQLAAKKYLNTIKNKRY 88. HSDAVFTDNYTRLRKQMAAHKYLNSIKNKRY 89. HSDAVFTDNYTRLRKQMAAKHYLNSIKNKRY 90. HSDAVFTDQYTRLRKQLAAHKYLNTIKNKRY 91. HSDAVFTDQYTRLRKQLAAKHYLNTIKNKRY 92. HSDAVFTDNYTRLRKQVAAKKYLQSIKKKR 93. HSDAVFTDNYTRLRKQVAAKKYLNSIKKKR 94. HSDAVFTDNYTRLRKQVAAKKYLNSIKNKRY 95. HSDAVFTDNYTRLRKQVAVKKYLQSIKKKR 96. HSDAVFTDNYTRLRKQVAVKKYLQSIKKK 97. HSDAVFTDNYTRLRKQVAVKKYLQSIKNKRY 98. HSDAVFTDNYTRLRKQVAAKKYLQSILKKRY 99. HSDAVFTDNYTRLRKQVAAKKYLQSILKKR 100. HSDAVFTDNYTRLRKQVAAKKYLQSILKK 101. HSDAVFTDNYTRLRKQVAAKKYLQSIKNK 102. HSDAVFTDNYTRLRKQVAVKKYLQSILKKRY 103. HSDAVFTDNYTRLRKQVAVKKYLQSILKKR 104. HSDAVFTDNYTRLRKQVAVKKYLQSILKK 105. HSDAVFTDNYTRLRKQVAVKKYLQSIKNK 106. HSDAVFTDNYTRLRKQVAAKKYLQSILNKRY 107. HSDAVFTDNYTRLRKQVAAKKYLQSILNKR 108. HSDAVFTDNYTRLRKQVAAKKYLQSILNK 109. HSDAVFTDNYTRLRKQMAEKKYLNSIKNKR 110. HSDAVFTDNYTRLRKQMAFKKYLNSIKNKR 111. HSDAVFTDNYTRLRKQMAGKKYLNSIKNKR 112. HSDAVFTDNYTRLRKQMAHKKYLNSIKNKR 113. HSDAVFTDNYTRLRKQMAIKKYLNSIKNKR 114. HSDAVFTDNYTRLRKQMAKKKYLNSIKNKR 115. HSDAVFTDNYTRLRKQMALKKYLNSIKNKR 116. HSDAVFTDNYTRLRKQMAMKKYLNSIKNKR 117. HSDAVFTDNYTRLRKQMANKKYLNSIKNKR 118. HSDAVFTDNYTRLRKQMAPKKYLNSIKNKR 119. HSDAVFTDNYTRLRKQMAQKKYLNSIKNKR 120. HSDAVFTDNYTRLRKQMARKKYLNSIKNKR 121. HSDAVFTDNYTRLRKQMASKKYLNSIKNKR 122. HSDAVFTDNYTRLRKQMATKKYLNSIKNKR 123. HSDAVFTDNYTRLRKQMAVKKYLNSIKNKR 124. HSDAVFTDNYTRLRKQMAWKKYLNSIKNKR 125. HSDAVFTDNYTRLRKQMAYKKYLNSIKNKR 126. HSDAVFTDNYTRLRKQMAAKKYLNSIANKR 127. HSDAVFTDNYTRLRKQMAAKKYLNSIDNKR 128. HSDAVFTDNYTRLRKQMAAKKYLNSIENKR 129. HSDAVFTDNYTRLRKQMAAKKYLNSIFNKR 130. HSDAVFTDNYTRLRKQMAAKKYLNSIGNKR 131. HSDAVFTDNYTRLRKQMAAKKYLNSIHNKR 132. HSDAVFTDNYTRLRKQMAAKKYLNSIINKR 133. HSDAVFTDNYTRLRKQMAAKKYLNSIMNKR 134. HSDAVFTDNYTRLRKQMAAKKYLNSINNKR 135. HSDAVFTDNYTRLRKQMAAKKYLNSIPNKR 136. HSDAVFTDNYTRLRKQMAAKKYLNSIQNKR 137. HSDAVFTDNYTRLRKQMAAKKYLNSIRNKR 138. HSDAVFTDNYTRLRKQMAAKKYLNSISNKR 139. HSDAVFTDNYTRLRKQMAAKKYLNSITNKR 140. HSDAVFTDNYTRLRKQMAAKKYLNSIVNKR 141. HSDAVFTDNYTRLRKQMAAKKYLNSIWNKR 142. HSDAVFTDNYTRLRKQMAAKKYLNSIYNKR 143. HSDAVFTDNYTRLRKQMAAKKYLNSIKNAR 144. HSDAVFTDNYTRLRKQMAAKKYLNSIKNDR 145. HSDAVFTDNYTRLRKQMAAKKYLNSIKNER 146. HSDAVFTDNYTRLRKQMAAKKYLNSIKNFR 147. HSDAVFTDNYTRLRKQMAAKKYLNSIKNGR 148. HSDAVFTDNYTRLRKQMAAKKYLNSIKNHR 149. HSDAVFTDNYTRLRKQMAAKKYLNSIKNIR 150. HSDAVFTDNYTRLRKQMAAKKYLNSIKNLR 151. HSDAVFTDNYTRLRKQMAAKKYLNSIKNMR 152. HSDAVFTDNYTRLRKQMAAKKYLNSIKNNR 153. HSDAVFTDNYTRLRKQMAAKKYLNSIKNPR 154. HSDAVFTDNYTRLRKQMAAKKYLNSIKNQR 155. HSDAVFTDNYTRLRKQMAAKKYLNSIKNRR 156. HSDAVFTDNYTRLRKQMAAKKYLNSIKNSR 157. HSDAVFTDNYTRLRKQMAAKKYLNSIKNTR 158. HSDAVFTDNYTRLRKQMAAKKYLNSIKNVR 159. HSDAVFTDNYTRLRKQMAAKKYLNSIKNWR 160. HSDAVFTDNYTRLRKQMAAKKYLNSIKNYR 161. HSDAVFTDNYTRLRKQMAAKKYLNSIKNKA 162. HSDAVFTDNYTRLRKQMAAKKYLNSIKNKD 163. HSDAVFTDNYTRLRKQMAAKKYLNSIKNKE 164. HSDAVFTDNYTRLRKQMAAKKYLNSIKNKF 165. HSDAVFTDNYTRLRKQMAAKKYLNSIKNKG 166. HSDAVFTDNYTRLRKQMAAKKYLNSIKNKH 167. HSDAVFTDNYTRLRKQMAAKKYLNSIKNKI 168. HSDAVFTDNYTRLRKQMAAKKYLNSIKNKK 169. HSDAVFTDNYTRLRKQMAAKKYLNSIKNKL 170. HSDAVFTDNYTRLRKQMAAKKYLNSIKNKM 171. HSDAVFTDNYTRLRKQMAAKKYLNSIKNKN 172. HSDAVFTDNYTRLRKQMAAKKYLNSIKNKP 173. HSDAVFTDNYTRLRKQMAAKKYLNSIKNKQ 174. HSDAVFTDNYTRLRKQMAAKKYLNSIKNKS 175. HSDAVFTDNYTRLRKQMAAKKYLNSIKNKT 176. HSDAVFTDNYTRLRKQMAAKKYLNSIKNKV 177. HSDAVFTDNYTRLRKQMAAKKYLNSIKNKW 178. HSDAVFTDNYTRLRKQMAAKKYLNSIKNKY 179. HSDAVFTDNYTRLRKQVAAKKYLQSIKNKRYSWCEPGWCR 180. HSDAVFTDDYTRLRKEVAAKKYLESIKDKRY 181. HSDAVFTDNYTRLRKQMAAKKYLNSIKNRI 182. HSDAVFTDNYTRLRKQMAGKKYLNSIKNRI 183. HSDAVFTDNYTRLRKQMAKKKYLNSIKNRI 184. HSDAVFTDNYTRLRKQMARKKYLNSIKNRI 185. HSDAVFTDNYTRLRKQMASKKYLNSIKNRI 186. HSDAVFTDNYTRLRKQMAAKKYLNSIPNRI 187. HSDAVFTDNYTRLRKQMAGKKYLNSIPNRI 188. HSDAVFTDNYTRLRKQMAKKKYLNSIPNRI 189. HSDAVFTDNYTRLRKQMARKKYLNSIPNRI 190. HSDAVFTDNYTRLRKQMASKKYLNSIPNRI 191. HSDAVFTDNYTRLRKQMAAKKYLNSIQNRI 192. HSDAVFTDNYTRLRKQMAGKKYLNSIQNRI 193. HSDAVFTDNYTRLRKQMAKKKYLNSIQNRI 194. HSDAVFTDNYTRLRKQMARKKYLNSIQNRI 195. HSDAVFTDNYTRLRKQMASKKYLNSIQNRI 196. HSDAVFTDNYTRLRKQMAAKKYLNSIRNRI 197. HSDAVFTDNYTRLRKQMAGKKYLNSIRNRI 198. HSDAVFTDNYTRLRKQMAKKKYLNSIRNRI 199. HSDAVFTDNYTRLRKQMARKKYLNSIRNRI 200. HSDAVFTDNYTRLRKQMASKKYLNSIRNRI 201. HSDAVFTENYTKLRKQLAAKKYLNDLKKGGT-NH₂ 202. HSDAVFTENYTKLRKQLAAKKYLNDLKKGGT 203. HSDAVFTENYTKLRKQLAAKKYLNDLKKGGT 204. HSDAVFTENYTKLRKQLAAKKYLNDLKK 205. HSDAVFTDNYTRLRKQLAAKKYLNDIKKGGT 206. HSDAVFTDNYTRLRKQLAAKKYLNDIKK-NH₂ 207. HSDAVFTDNYTRLRKQMAVKKYLNDLKKGGT 208. HSDAVFTDNYTRLRKQMAAKKYLNDIKKGGT 209. HSDAVFTDNYTRLRKQLAVKKYLNDIKKGGT 210. HSDAVFTDNYTRLRKQLAAKKYLNDIKKGG 211. HSDAVFTDNYTRLRKQLAAKKYLNDIKKG 212. HSDAVFTDNYTRLRKQLAAKKYLNDIKK 213. HSDAVFTDNYTRLRKQLAAKKYLNDIKKQ 214. HSDAVFTDNYTRLRKQLAAKKYLNDIKKNQ 215. HSDAVFTDNYTRLREQMAVKKYLNSILN 216. HSDAVFTDNYTRLRKQLAVKKYLNSILN 217. HSDAVFTDNYTRLRKQMAAKKYLNSILN 218. HSDAVFTENYTKLRKQLAAKKYLNDLKKGGT 219. HSDAVFTDNYTRLRKQMACKKYLNSIKNKR 220. HSDAVFTDNYTRLRKQMADKKYLNSIKNKR 221. HSDAVFTDNYTRLRKQMAAKKYLNSICNKR 222. HSDAVFTDNYTRLRKQMAAKKYLNSIKNCR 223. HSDAVFTDQYTRLRKQVAAKKYLQSIKQKRY 224. HTDAVFTDQYTRLRKQVAAKKYLQSIKQKRY 225. HSDAVFTDQYTRLRKQMAAKKYLQSIKQKRY 226. HSDAVFTDQYTRLRKQVAAKKYLQSIKQK 227. HTEAVFTDQYTRLRKQVAAKKYLQSIKQKRY 228. HSDAVFTDQYTRLRKQLAVKKYLQDIKQGGT 229. HSDAVFTDQYTRLRKQMAAKKYLQSIKQKR 230. HSDAVFTDQYTRLRKQLAAKKYLQTIKQKRY 231. HSDAVFTDQYTRLRKQMAAKKYLQTIKQKRY 232. HSDAVFTDQYTRLRKQMAAHKYLQSIKQKRY 233. HSDAVFTDQYTRLRKQMAAKHYLQSIKQKRY 234. HSDAVFTDQYTRLRKQMAGKKYLQSIKQKR 235. HSDAVFTDQYTRLRKQMAKKKYLQSIKQKR 236. HSDAVFTDQYTRLRKQMARKKYLQSIKQKR 237. HSDAVFTDQYTRLRKQMASKKYLQSIKQKR 238. HSDAVFTDQYTRLRKQMAAKKYLQSIPQKR 239. HSDAVFTDQYTRLRKQMAAKKYLQSIQQKR 240. HSDAVFTDQYTRLRKQMAAKKYLQSIRQKR 241. HSDAVFTDQYTRLRKQMAAKKYLQSIKQRR 242. HSDAVFTDQYTRLRKQMAAKKYLQSIKQKA 243. HSDAVFTDQYTRLRKQMAAKKYLQSIKQKF 244. HSDAVFTDQYTRLRKQMAAKKYLQSIKQKH 245. HSDAVFTDQYTRLRKQMAAKKYLQSIKQKI 246. HSDAVFTDQYTRLRKQMAAKKYLQSIKQKK 247. HSDAVFTDQYTRLRKQMAAKKYLQSIKQKL 248. HSDAVFTDQYTRLRKQMAAKKYLQSIKQKM 249. HSDAVFTDQYTRLRKQMAAKKYLQSIKQKP 250. HSDAVFTDQYTRLRKQMAAKKYLQSIKQKQ 251. HSDAVFTDQYTRLRKQMAAKKYLQSIKQKS 252. HSDAVFTDQYTRLRKQMAAKKYLQSIKQKT 253. HSDAVFTDQYTRLRKQMAAKKYLQSIKQKV 254. HSDAVFTDQYTRLRKQMAAKKYLQSIKQKW 255. HSDAVFTDQYTRLRKQMAAKKYLQSIKQKY 256. HSDAVFTDQYTRLRKQMAGKKYLQSIKQRI 257. HSDAVFTDQYTRLRKQMAKKKYLQSIKQRI 258. HSDAVFTDQYTRLRKQMASKKYLQSIKQRI 259. HSDAVFTDQYTRLRKQMAAKKYLQSIPQRI 260. HSDAVFTDQYTRLRKQMASKKYLQSIRQRI 261. HSDAVFTDNYTRLRKQVAAKKYLQSIKQKRY 262. HTDAVFTDNYTRLRKQVAAKKYLQSIKQKRY 263. HSDAVFTDNYTRLRKQMAAKKYLQSIKQKRY 264. HSDAVFTDNYTRLRKQVAAKKYLQSIKQK 265. HTEAVFTDNYTRLRKQVAAKKYLQSIKQKRY 266. HSDAVFTDNYTRLRKQLAVKKYLQDIKQGGT 267. HSDAVFTDNYTRLRKQMAAKKYLQSIKQKR 268. HSDAVFTDNYTRLRKQLAAKKYLQTIKQKRY 269. HSDAVFTDNYTRLRKQMAAKKYLQTIKQKRY 270. HSDAVFTDNYTRLRKQMAAHKYLQSIKQKRY 271. HSDAVFTDNYTRLRKQMAAKHYLQSIKQKRY 272. HSDAVFTDNYTRLRKQMAGKKYLQSIKQKR 273. HSDAVFTDNYTRLRKQMAKKKYLQSIKQKR 274. HSDAVFTDNYTRLRKQMARKKYLQSIKQKR 275. HSDAVFTDNYTRLRKQMASKKYLQSIKQKR 276. HSDAVFTDNYTRLRKQMAAKKYLQSIPQKR 277. HSDAVFTDNYTRLRKQMAAKKYLQSIQQKR 278. HSDAVFTDNYTRLRKQMAAKKYLQSIRQKR 279. HSDAVFTDNYTRLRKQMAAKKYLQSIKQRR 280. HSDAVFTDNYTRLRKQMAAKKYLQSIKQKA 281. HSDAVFTDNYTRLRKQMAAKKYLQSIKQKF 282. HSDAVFTDNYTRLRKQMAAKKYLQSIKQKH 283. HSDAVFTDNYTRLRKQMAAKKYLQSIKQKI 284. HSDAVFTDNYTRLRKQMAAKKYLQSIKQKK 285. HSDAVFTDNYTRLRKQMAAKKYLQSIKQKL 286. HSDAVFTDNYTRLRKQMAAKKYLQSIKQKM 287. HSDAVFTDNYTRLRKQMAAKKYLQSIKQKP 288. HSDAVFTDNYTRLRKQMAAKKYLQSIKQKQ 289. HSDAVFTDNYTRLRKQMAAKKYLQSIKQKS 290. HSDAVFTDNYTRLRKQMAAKKYLQSIKQKT 291. HSDAVFTDNYTRLRKQMAAKKYLQSIKQKV 292. HSDAVFTDNYTRLRKQMAAKKYLQSIKQKW 293. HSDAVFTDNYTRLRKQMAAKKYLQSIKQKY 294. HSDAVFTDNYTRLRKQMAGKKYLQSIKQRI 295. HSDAVFTDNYTRLRKQMAKKKYLQSIKQRI 296. HSDAVFTDNYTRLRKQMASKKYLQSIKQRI 297. HSDAVFTDNYTRLRKQMAAKKYLQSIPQRI 298. HSDAVFTDNYTRLRKQMASKKYLQSIRQRI 299. HSDAVFTDQYTRLRKQVAAKKYLQSIKNKRY 300. HTDAVFTDQYTRLRKQVAAKKYLQSIKNKRY 301. HSDAVFTDQYTRLRKQMAAKKYLQSIKNKRY 302. HSDAVFTDQYTRLRKQVAAKKYLQSIKNK 303. HTEAVFTDQYTRLRKQVAAKKYLQSIKNKRY 304. HSDAVFTDQYTRLRKQLAVKKYLQDIKNGGT 305. HSDAVFTDQYTRLRKQMAAKKYLQSIKNKR 306. HSDAVFTDQYTRLRKQLAAKKYLQTIKNKRY 307. HSDAVFTDQYTRLRKQMAAKKYLQTIKNKRY 308. HSDAVFTDQYTRLRKQMAAHKYLQSIKNKRY 309. HSDAVFTDQYTRLRKQMAAKHYLQSIKNKRY 310. HSDAVFTDQYTRLRKQMAGKKYLQSIKNKR 311. HSDAVFTDQYTRLRKQMAKKKYLQSIKNKR 312. HSDAVFTDQYTRLRKQMARKKYLQSIKNKR 313. HSDAVFTDQYTRLRKQMASKKYLQSIKNKR 314. HSDAVFTDQYTRLRKQMAAKKYLQSIPNKR 315. HSDAVFTDQYTRLRKQMAAKKYLQSIQNKR 316. HSDAVFTDQYTRLRKQMAAKKYLQSIRNKR 317. HSDAVFTDQYTRLRKQMAAKKYLQSIKNRR 318. HSDAVFTDQYTRLRKQMAAKKYLQSIKNKA 319. HSDAVFTDQYTRLRKQMAAKKYLQSIKNKF 320. HSDAVFTDQYTRLRKQMAAKKYLQSIKNKH 321. HSDAVFTDQYTRLRKQMAAKKYLQSIKNKI 322. HSDAVFTDQYTRLRKQMAAKKYLQSIKNKK 323. HSDAVFTDQYTRLRKQMAAKKYLQSIKNKL 324. HSDAVFTDQYTRLRKQMAAKKYLQSIKNKM 325. HSDAVFTDQYTRLRKQMAAKKYLQSIKNKP 326. HSDAVFTDQYTRLRKQMAAKKYLQSIKNKQ 327. HSDAVFTDQYTRLRKQMAAKKYLQSIKNKS 328. HSDAVFTDQYTRLRKQMAAKKYLQSIKNKT 329. HSDAVFTDQYTRLRKQMAAKKYLQSIKNKV 330. HSDAVFTDQYTRLRKQMAAKKYLQSIKNKW 331. HSDAVFTDQYTRLRKQMAAKKYLQSIKNKY 332. HSDAVFTDQYTRLRKQMAGKKYLQSIKNRI 333. HSDAVFTDQYTRLRKQMAKKKYLQSIKNRI 334. HSDAVFTDQYTRLRKQMASKKYLQSIKNRI 335. HSDAVFTDQYTRLRKQMAAKKYLQSIPNRI 336. HSDAVFTDQYTRLRKQMASKKYLQSIRNRI 337. HSDAVFTDQYTRLRKQVAAKKYLQSIKQKRYC 338. HTDAVFTDQYTRLRKQVAAKKYLQSIKQKRYC 339. HSDAVFTDQYTRLRKQMAAKKYLQSIKQKRYC 340. HSDAVFTDQYTRLRKQVAAKKYLQSIKQKC 341. HTEAVFTDQYTRLRKQVAAKKYLQSIKQKRYC 342. HSDAVFTDQYTRLRKQLAVKKYLQDIKQGGTC 343. HSDAVFTDQYTRLRKQMAAKKYLQSIKQKRC 344. HSDAVFTDQYTRLRKQLAAKKYLQTIKQKRYC 345. HSDAVFTDQYTRLRKQMAAKKYLQTIKQKRYC 346. HSDAVFTDQYTRLRKQMAAHKYLQSIKQKRYC 347. HSDAVFTDQYTRLRKQMAAKHYLQSIKQKRYC 348. HSDAVFTDQYTRLRKQMAGKKYLQSIKQKRC 349. HSDAVFTDQYTRLRKQMAKKKYLQSIKQKRC 350. HSDAVFTDQYTRLRKQMARKKYLQSIKQKRC 351. HSDAVFTDQYTRLRKQMASKKYLQSIKQKRC 352. HSDAVFTDQYTRLRKQMAAKKYLQSIPQKRC 353. HSDAVFTDQYTRLRKQMAAKKYLQSIQQKRC 354. HSDAVFTDQYTRLRKQMAAKKYLQSIRQKRC 355. HSDAVFTDQYTRLRKQMAAKKYLQSIKQRRC 356. HSDAVFTDQYTRLRKQMAAKKYLQSIKQKAC 357. HSDAVFTDQYTRLRKQMAAKKYLQSIKQKFC 358. HSDAVFTDQYTRLRKQMAAKKYLQSIKQKHC 359. HSDAVFTDQYTRLRKQMAAKKYLQSIKQKIC 360. HSDAVFTDQYTRLRKQMAAKKYLQSIKQKKC 361. HSDAVFTDQYTRLRKQMAAKKYLQSIKQKLC 362. HSDAVFTDQYTRLRKQMAAKKYLQSIKQKMC 363. HSDAVFTDQYTRLRKQMAAKKYLQSIKQKPC 364. HSDAVFTDQYTRLRKQMAAKKYLQSIKQKQC 365. HSDAVFTDQYTRLRKQMAAKKYLQSIKQKSC 366. HSDAVFTDQYTRLRKQMAAKKYLQSIKQKTC 367. HSDAVFTDQYTRLRKQMAAKKYLQSIKQKVC 368. HSDAVFTDQYTRLRKQMAAKKYLQSIKQKWC 369. HSDAVFTDQYTRLRKQMAAKKYLQSIKQKYC 370. HSDAVFTDQYTRLRKQMAGKKYLQSIKQRIC 371. HSDAVFTDQYTRLRKQMAKKKYLQSIKQRIC 372. HSDAVFTDQYTRLRKQMASKKYLQSIKQRIC 373. HSDAVFTDQYTRLRKQMAAKKYLQSIPQRIC 374. HSDAVFTDQYTRLRKQMASKKYLQSIRQRIC

Another alternative preferred sequence for selective PEGylated VPAC2 receptor peptide agonists of the present invention comprises an amino acid sequence of the Formula 4 (SEQ ID NO: 7), provided that if Xaa₂₉, Xaa₃₀, Xaa₃₁, Xaa₃₂, Xaa₃₃, Xaa₃₄, Xaa₃₅, Xaa₃₆, Xaa₃₇, Xaa₃₈, or Xaa₃₉ of Formula 4 (SEQ ID NO: 7) is absent, the next amino acid present downstream is the next amino acid in the peptide sequence and wherein the C-terminal amino acid may be amidated. For example, if Xaa₂₉ is Lys and Xaa₃₀ is absent, the next amino acid bonded to Lys at position 29 is an amino acid listed for position 31 or, if position 31 is also absent, an amino acid listed for position 32 is bonded to Lys at position 29, and so forth. Additionally, for example, if Xaa₂₉ is Lys and Xaa₃₀ through Xaa₄₀ are absent, Lys may be the C-terminal amino acid and may be amidated.

Preferably, an alternative selective PEGylated VPAC2 receptor peptide agonist of the present invention has the amino acid sequence of Formula 4 (SEQ ID NO: 7), modified so that from one, two, three, four, five, six, seven, eight, nine, or ten amino acids differ from the amino acid in the corresponding position of SEQ ID NO: 1.

Preferably, the C-terminal extension for an alternative embodiment of the present invention comprises an amino acid sequence of the Formula 5 (SEQ ID NO: 8), provided that if Xaa₁, Xaa₂, Xaa₃, Xaa₄, Xaa₅, or Xaa₆ of Formula 5 (SEQ ID NO: 8) is absent, the next amino acid present downstream is the next amino acid in the C-terminal extension and wherein the C-terminal amino acid may be amidated. For example, if Xaa₁ is Ser and Xaa₂ is absent, the next amino acid bonded to Ser at position 1 is an amino position 3 or, if position 3 is also absent, an amino acid listed for position Ser at position 1, and so forth. Additionally, for example, if Xaa₁ is Ser and Xaa₂ through Xaa₁₃ are absent, Ser may be the C-terminal amino acid and may be amidated.

More preferably, the C-terminal extension of an alternative embodiment of the present invention includes the following sequences and variants thereof:

SEQ ID # Sequence SEQ ID NO: 9 SRTSPPP SEQ ID NO: 10 SRTSPPP-NH₂

Preferably, the C-terminal extension differs from SEQ ID NO: 9, or SEQ ID NO: 10, by no more than six amino acids, more preferably by no more than five amino acids, even more preferably by no more than four amino acids, still more preferably by no more than three amino acids, yet more preferably by no more than two amino acids, and most preferably by no more than one amino acid.

These sequences contain the standard single letter codes for the twenty naturally occurring amino acids. SEQ ID NO: 10 contains a sequence that is amidated at the C-terminus of the sequence.

Another alternative preferred C-terminal extension of the present invention comprises an amino acid sequence of the Formula 6 (SEQ ID NO: 11), provided that if Xaa₁, Xaa₂, Xaa₃, Xaa₄, Xaa₅, Xaa₆, Xaa₇, or Xaa₈ of Formula 6 (SEQ ID NO: 11) is absent, the next amino acid present downstream is the next amino acid in the C-terminal extension and wherein the C-terminal amino acid may be amidated. For example, if Xaa₁ is Ser and Xaa₂ is absent, the next amino acid bonded to Ser at position 1 is an amino acid listed for position 3 or, if position 3 is also absent, an amino acid listed for position 4 is bonded to Ser at position 1, and so forth. Additionally, for example, if Xaa₁ is Ser and Xaa₂ through Xaa₉ are absent, Ser may be the C-terminal amino acid and may be amidated.

Another alternative preferred C-terminal extension of the present invention includes (Lys)_(n) or (Glu)_(n) wherein n is the number of lysine or glutamic acid residues added to the C-terminus and wherein n can be anywhere from one to eight residues.

PEGylation of proteins may overcome many of the pharmacological and toxicological/immunological problems associated with using peptides and therapeutics. However, for any individual peptide it is uncertain whether form of the peptide will have significant loss in bioactivity as compared to the unPEGylated form of the peptide.

The bioactivity of PEGylated proteins can be affected by factors such as: i) the size of the PEG molecule; ii) the particular sites of attachment; iii) the degree of modification; iv) adverse coupling conditions; v) whether a linker is used for attachment or whether the polymer is directly attached; vi) generation of harmful co-products; vii) damage inflicted by the activated polymer; or viii) retention of charge. Work performed on the PEGylation of cytokines, for example, shows the effect PEGylation may have. Depending on the coupling reaction used, polymer modification of cytokines has resulted in dramatic reductions in bioactivity. [Francis, G. E., et al., (1998) PEGylation of cytokines and other therapeutic proteins and peptides: the importance of biological optimization of coupling techniques, Intl. J. Hem. 68:1-18]. Maintaining the bioactivity of PEGylated peptides is even more problematic than for proteins. As peptides are smaller than proteins, modification by PEGylation may potentially have a greater effect on bioactivity.

The VPAC2 receptor peptide agonists of the present invention are modified by the covalent attachment of one or more molecules of a polyethylene glycol (PEG) and generally have improved pharmacokinetic profiles due to slower proteolytic degradation and renal clearance. Attachment of PEG molecule(s) (PEGylation) will increase the apparent size of the VPAC2 receptor peptide agonists, thus reducing renal filtration and altering biodistribution. PEGylation can shield antigenic epitopes of the VPAC2 receptor peptide agonists, thus reducing reticuloendothelial clearance and recognition by the immune system and also reducing degradation by proteolytic enzymes, such as DPP-IV.

Covalent attachment of one or more molecules of polyethylene glycol to a small, biologically active VPAC2 receptor peptide agonist poses the risk of adversely affecting the agonist, for example, by destabilising the inherent secondary structure and bioactive conformation and reducing bioactivity, so as to make the agonist unsuitable for use as a therapeutic. The present invention, however, is based on the finding that covalent attachment of one or more molecules of PEG to particular residues of a VPAC2 receptor peptide agonist surprisingly results in a biologically active, PEGylated V peptide agonist with an extended half-life and reduced clearance when of non PEGylated VPAC2 receptor peptide agonists. The compounds of the present invention include selective PEGylated VPAC2 receptor peptide agonists.

In order to determine the potential PEGylation sites in a VPAC2 receptor peptide agonist, serine scanning may be conducted. A Ser residue is substituted at a particular position in the peptide and the Ser-modified peptide is tested for potency and selectivity. If the Ser substitution has minimal impact on potency and the Ser-modified peptide is selective for the VPAC2 receptor, the Ser residue is then substituted for a Cys or Lys residue, which serves as a direct or indirect PEGylation site. Indirect PEGylation of a residue is the PEGylation of a chemical group or residue which is bonded to the PEGylation site residue. Indirect PEGylation of Lys includes PEGylation of K(W) and K(CO(CH₂)₂SH).

The invention described herein provides VPAC2 receptor peptide agonists covalently attached to one or more molecules of polyethylene glycol (PEG), or a derivative thereof wherein each PEG is attached to a Cys or Lys amino acid, to a K(W) or a K(CO(CH₂)₂SH), or to the carboxy terminal amino acid of the peptide agonist. PEGylation can enhance the half-life of the selective VPAC2 receptor peptide agonists, resulting in PEGylated VPAC2 receptor peptide agonists with an elimination half-life of at least one hour, preferably at least 3, 5, 7, 10, 15, 20, or 24 hours and most preferably at least 48 hours. The PEGylated VPAC2 receptor peptide agonists of the present invention preferably have a clearance value of 200 ml/h/kg or less, more preferably 180, 150, 120, 100, 80, 60 ml/h/kg or less and most preferably less than 50, 40 or 20 ml/h/kg.

The present invention encompasses the discovery that specific amino acids added to the C-terminus of a peptide sequence for a PEGylated VPAC2 receptor peptide agonist provide features that may protect the peptide as well as may enhance activity, selectivity, and/or potency. For example, these C-terminal extensions may stabilize the helical structure of the peptide and sites within the peptide prone to enzymatic cleavage that are located near the C-terminus. Furthermore, many of the C-terminally extended peptides disclosed herein may be more selective for the VPAC2 receptor and can be more potent than VIP, PACAP, and other known VPAC2 receptor peptide agonists.

It has furthermore been discovered that N-terminal modification PEGylated VPAC2 receptor peptide agonist may enhance potency and against DPP-IV cleavage.

VIP and some known VPAC2 receptor peptide agonists are susceptible to cleavage by various enzymes and, thus, have a short in vivo half-life. Various enzymatic cleavage sites in the VPAC2 receptor peptide agonists are discussed below. The cleavage sites are discussed relative to the amino acid positions in VIP (SEQ ID NO: 1), and are applicable to the sequences noted herein.

Cleavage of the peptide agonist by the enzyme dipeptidyl-peptidase-IV (DPP-IV) occurs between position 2 (serine in VIP) and position 3 (aspartic acid in VIP). The addition of a N-terminal modification and/or various substitutions at position 2 may improve stability against DPP-IV cleavage. Examples of amino acids at position 2 that may improve stability against DPP-IV inactivation preferably include valine, D-alanine, or D-serine. More preferably, position 2 is valine or D-alanine. Examples of N-terminal modifications that may improve stability against DPP-IV inactivation include the addition of acetyl, propionyl, butyryl, pentanoyl, hexanoyl, methionine, methionine sulfoxide, 3-phenylpropionyl, phenylacetyl, benzoyl, norleucine, D-histidine, isoleucine, 3-mercaptopropionyl, biotinyl-6-aminohexanoic acid and —C(═O—NH)—NH₂. Preferably, the N-terminal modification is the addition of acetyl or hexanoyl.

There are chymotrypsin cleavage sites in wild-type VIP between the amino acids 10 and 11 (tyrosine and threonine) and those at 22 and 23 (tyrosine and leucine). Making substitutions at position 10 and/or 11 and position 22 and/or 23 may increase the stability of the peptide at these sites.

There is also a trypsin cleavage site between arginine at position 12 and leucine at position 13 of wild-type VIP. Examples of substitutions which render the peptide resistant to cleavage by trypsin at this site include substitution of the arginine at position 12 with ornithine and substitution of leucine at position 13 with amino isobutyric acid.

In wild-type VIP, and in numerous VPAC2 receptor peptide agonists known in the art, there are cleavage sites between the basic amino acids at positions 14 and 15 and between those at positions 20 and 21. The selective VPAC2 receptor agonists of the present invention generally have improved proteolytic stability in vivo due to substitutions in these sites. These substitutions can render the peptide resistant to cleavage by trypsin-like enzymes, including trypsin. Examples of amino acids at position 14 that confer some resistance to cleavage by trypsin-like enzymes alone or in combination with the amino acids specified for position 15 below include glutamine, amino isobutyric acid, homoarginine, ornithine, citrulline, lysine, alanine and leucine. Also, position 14 may be arginine when position 15 is an amino acid other than lysine. Also, position 14 can be arginine when position 15 is lysine, but this specific combination does not address enzymatic cleavage. Examples of amino acids at position 15 that confer some resistance to cleavage by trypsin-like enzymes alone or in combination with amino acids specified above for position 14 include amino isobutyric acid, ornithine and arginine. Also, position 15 may be lysine when position 14 is an amino acid other than arginine. Also, position 15 can be lysine when position 14 is arginine, but this specific combination does not address enzymatic cleavage. Examples of amino acids at position 20 that confer some resistance to cleavage by trypsin-like enzymes alone or in combination with amino acids specified for position 21 include valine, leucine, amino isobutyric acid, alanine, glutamine and arginine. Also, position 20 may be lysine when position 21 is an amino acid other than lysine. Also, position 20 can be lysine when position 21 is lysine, but this specific combination does not address enzymatic cleavage. An example of an amino acid at position 21 that confers some resistance to cleavage by trypsin-like peptides alone or in combination with amino acids specified for position 20 include amino isobutyric acid, ornithine, alanine, glutamine or arginine. Also, position 21 may be lysine when position 20 is an amino acid other than lysine. Also, position 21 can be lysine when position 20 is lysine, but this specific combination does not address enzymatic cleavage. The improved stability of a representative number of selective PEGylated VPAC2 receptor peptide agonists with resistance to peptidase cleavage and encompassed by the present invention is demonstrated in Example 6.

The bond between the amino acids at positions 25 and 26 of wild-type VIP is susceptible to enzymatic cleavage. This cleavage site can be completely or partially eliminated through substitution of the amino acid at position 25 and/or the amino acid at position 26. Examples of amino acids at position 25 that confer at least some resistance to enzymatic cleavage include phenylalanine, isoleucine, leucine, threonine, valine, tryptophan, glutamine, asparagine, tyrosine, or amino isobutyric acid. Also, position 25 may be serine when position 26 is an amino acid other than isoleucine. Also, position 25 can be serine when position 26 is isoleucine, but this specific combination does not address enzymatic cleavage. Examples of amino acids at positions 26 that confer at least some resistance to enzymatic cleavage alone or in combination with the amino acids specified above for position 25 include leucine, threonine, valine, tryptophan, tyrosine, phenylalanine, or amino isobutyric acid. Also, position 26 may be isoleucine when position 25 is an amino acid other than serine. Also, position 26 can be isoleucine when position 25 is serine, but this specific combination does not address enzymatic cleavage.

In addition to selective VPAC2 receptor peptide agonists with resistance to cleavage by various peptidases, the selective PEGylated VPAC2 peptide receptor agonists of the present invention may also encompass peptides with enhanced selectivity for the VPAC2 receptor, increased potency, and/or increased stability compared with some peptides known in the art. Examples of amino acid positions that may affect such properties include positions: 3, 8, 9, 12, 14, 15, 16, 17, 20, 21, 27, 28, and 29 of Formula 7, 9, and 11. For example, the amino acid at position 3 is preferably aspartic acid or glutamic acid; the amino acid at position 8 is preferably aspartic acid or glutamic acid; the amino acid at position 9 is preferably asparagine or glutamine; the amino acid at position 12 is preferably arginine, homoarginine, ornithine, or lysine; the amino acid at position 14 is preferably arginine, glutamine, amino isobutyric acid, homoarginine, ornithine, citrulline, lysine, alanine, or leucine; the amino acid at position 15 is preferably lysine, leucine, amino isobutyric acid, ornithine or arginine; the amino acid at position 16 is preferably glutamine or lysine; the amino acid at position 17 is preferably valine, alanine, leucine, isoleucine, lysine, or norleucine; the amino acid at position 20 is preferably lysine, valine, leucine, amino isobutyric acid, alanine, glutamine or arginine; the amino acid at position 21 is preferably lysine, amino isobutyric acid, ornithine, alanine, glutamine or arginine; the amino acid at position 27 is preferably lysine, ornithine, homoarginine or arginine; the amino acid at position 28 is preferably asparagine, glutamine, lysine, homoarginine, amino isobutyric acid, proline, or ornithine; and, if present, the amino acid at position 29 is preferably lysine, ornithine, or homoarginine. Preferably, these amino acid substitutions may be combined with substitutions at positions that affect the four aforementioned regions susceptible to cleavage by various enzymes.

The increased potency and selectivity for various VPAC2 receptor peptide agonists of the present invention is demonstrated in Examples 3 and 4. For example, Table 1 in Example 3 provides a list of selective PEGylated VPAC2 receptor peptide agonists and their corresponding in vitro potency results. Preferably, the selective PEGylated VPAC2 receptor peptide agonists of the present invention have an EC₅₀ value less than 200 nM. More preferably, the EC₅₀ value is less than 50 nM. Even more preferably, the EC₅₀ value is less than 30 nM. Still more preferably, the EC₅₀ value is less than 10 nM.

Table 2 in Example 4 provides a list of PEGylated VPAC2 receptor peptide agonists and their corresponding selectivity results for human VPAC2, VPAC1, and PAC1. See Example 4 for further details of these assays. These results are provided as a ratio of VPAC2 binding affinity to VPAC1 binding affinity and as a ratio of VPAC2 binding affinity to PAC1 binding affinity. Preferably, the agonists of the present invention have a selectivity ratio where the affinity for VPAC2 is at least 50 times greater than for VPAC1 and/or for PAC1. More preferably, the affinity is at least 100 times greater than for VPAC1 and/or for PAC1. Even more preferably, the affinity is at least 200 times greater than for VPAC1 and/or for PAC1. Still more preferably, the affinity is at least 500 times greater than for VPAC1 and/or for PAC1. Yet more preferably, the affinity is at least 1000 times greater than for VPAC1 and/or for PAC1.

As used herein, selective PEGylated VPAC2 receptor peptide agonists also include pharmaceutically acceptable salts of the compounds described herein. A selective PEGylated VPAC2 receptor peptide agonist of this invention can possess a sufficiently acidic, a sufficiently basic, or both functional groups, and accordingly react with any of a number of inorganic bases, and inorganic and organic acids, to form a salt. Acids commonly employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, trifluoroacetic acid, and the like. Examples of such salts include the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like.

Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Such bases useful in preparing the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, and the like.

The selective PEGylated VPAC2 receptor peptide agonists of the present invention can be administered parenterally. Parenteral administration can include, for example, systemic administration, such as by intramuscular, intravenous, subcutaneous, intradermal, or intraperitoneal injection. These agonists can be administered to the subject in conjunction with an acceptable pharmaceutical carrier, diluent, or excipient as part of a pharmaceutical composition for treating NIDDM, or the disorders discussed below. The pharmaceutical composition can be a solution or, if administered parenterally, a suspension of the VPAC2 receptor peptide agonist or a suspension of the VPAC2 receptor peptide agonist complexed with a divalent metal cation such as zinc. Suitable pharmaceutical carriers may contain inert ingredients which do not interact with the peptide or peptide derivative. Suitable pharmaceutical carriers for parenteral administration include, for example, sterile water, physiological saline, bacteriostatic saline (saline containing about 0.9% mg/ml benzyl alcohol), phosphate-buffered saline, Hank's solution, Ringer's-lactate and the like. Some examples of suitable excipients include lactose, dextrose, sucrose, trehalose, sorbitol, and mannitol.

Standard pharmaceutical formulation techniques may be employed such as those described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. The selective PEGylated VPAC2 receptor peptide agonists of the present invention may be formulated for administration through the buccal, topical, oral, transdermal, nasal, or pulmonary route.

The PEGylated VPAC2 receptor peptide agonists of the invention may be formulated for administration such that blood plasma levels are maintained in the efficacious range for extended time periods. The main barrier to effective oral peptide drug delivery is poor bioavailability due to degradation of peptides by acids and enzymes, poor absorption through epithelial membranes, and transition of peptides to an insoluble form after exposure to the acidic pH environment in the digestive tract. Oral delivery systems for peptides such as those encompassed by the present invention are known in the art. For example, PEGylated VPAC2 receptor peptide agonists can be encapsulated using microspheres and then delivered orally. For example, PEGylated VPAC2 receptor peptide agonists can be encapsulated into microspheres composed of a commercially available, biocompatible, biodegradable polymer, poly(lactide-co-glycolide)-COOH and olive oil as a filler (see Joseph, et al. Diabetologia 43:1319-1328 (2000)). Other types of microsphere technology is also available commercially such as Medisorb® and Prolease® biodegradable polymers from Alkermes. Medisorb® polymers can be produced with any of the lactide isomers. Lactide:glycolide ratios can be varied between 0:100 and 100:0 allowing for a broad range of polymer properties. This allows for the design of delivery systems and implantable devices with resorption times ranging from weeks to months. Emisphere has also published numerous articles discussing oral delivery technology for peptides and proteins. For example, see WO 95/28838 by Leone-bay et al. which discloses specific carriers comprised of modified amino acids to facilitate absorption.

The selective PEGylated VPAC2 receptor peptide agonists described herein can be used to treat subjects with a wide variety of diseases and conditions. Agonists encompassed by the present invention exert their biological effects by acting at a receptor referred to as the VPAC2 receptor. Subjects with diseases and/or conditions that respond favourably to VPAC2 receptor stimulation or to the administration of VPAC2 receptor peptide agonists can therefore be treated with the VPAC2 agonists of the present invention. These subjects are said to “be in need of treatment with VPAC2 agonists” or “in need of VPAC2 receptor stimulation”.

The selective PEGylated VPAC2 receptor peptide agonists of the present invention may be employed to treat diabetes, including both type 1 and type 2 diabetes (non-insulin dependent diabetes mellitus or NIDDM). Also included are subjects requiring prophylactic treatment with a VPAC2 receptor agonist, e.g., subjects at risk for developing NIDDM. Such treatment may also delay the onset of diabetes and diabetic complications. Additional subjects include those with impaired glucose tolerance or impaired fasting glucose, subjects whose body weight is about 25% above normal body weight for the subject's height and body build, subjects having one or more parents with NIDDM, subjects who have had gestational diabetes, and subjects with metabolic disorders such as those resulting from decreased endogenous insulin secretion. The selective PEGylated VPAC2 receptor peptide agonists may be used to prevent subjects with impaired glucose tolerance from proceeding to develop type 2 diabetes, prevent pancreatic β-cell deterioration, induce β-cell proliferation, improve β-cell function, activate dormant β-cells, differentiate cells into β-cells, stimulate β-cell replication, and inhibit β-cell apoptosis. Other diseases and conditions that may be treated or prevented using compounds of the invention in methods of the invention include: Maturity-Onset Diabetes of the Young (MODY) (Herman, et al., Diabetes 43:40, 1994); Latent Autoimmune Diabetes Adult (LADA) (Zimmet, et al., Diabetes Med. 11:299, 1994); impaired glucose tolerance (IGT) (Expert Committee on Classification of Diabetes Mellitus, Diabetes Care 22 (Supp. 1):S5, 1999); impaired fasting glucose (IFG) (Charles, et al., Diabetes 40:796, 1991); gestational diabetes (Metzger, Diabetes, 40:197, 1991); metabolic syndrome X, dyslipidemia, hyperglycemia, hyperinsulinemia, hypertriglyceridemia, and insulin resistance.

The selective PEGylated VPAC2 receptor peptide agonists of the invention may also be used in methods of the invention to treat secondary causes of diabetes (Expert Committee on Classification of Diabetes Mellitus, Diabetes Care 22 (Supp. 1):S5, 1999). Such secondary causes include glucocorticoid excess, growth hormone excess, pheochromocytoma, and drug-induced diabetes. Drugs that may induce diabetes include, but are not limited to, pyriminil, nicotinic acid, glucocorticoids, phenyloin, thyroid hormone, β-adrenergic agents, α-interferon and drugs used to treat HIV infection.

The selective PEGylated VPAC2 receptor peptide agonists of the present invention may be effective in the suppression of food intake and the treatment of obesity.

The selective PEGylated VPAC2 receptor peptide agonists of the present invention may also be effective in the prevention or treatment of such disorders as atherosclerotic disease, hyperlipidemia, hypercholesteremia, low HDL levels, hypertension, primary pulmonary hypertension, cardiovascular disease (including artereosclerosis, coronary heart disease, coronary artery disease, and hypertension), cerebrovascular disease and peripheral vessel disease; and for the treatment of lupus, polycystic ovary syndrome, carcinogenesis, and hyperplasia, asthma, male and female reproduction problems, sexual disorders, ulcers, sleep disorders, disorders of lipid and carbohydrate metabolism, circadian dysfunction, growth disorders, disorders of energy homeostasis, immune diseases including autoimmune diseases (e.g., systemic lupus erythematosus), as well as acute and chronic inflammatory diseases, rheumatoid arthritis, and septic shock.

The selective PEGylated VPAC2 receptor peptide agonists of the present invention may also be useful for treating physiological disorders related to, for example, cell differentiation to produce lipid accumulating cells, regulation of insulin sensitivity and blood glucose levels, which are involved in, for example, abnormal pancreatic β-cell function, insulin secreting tumors and/or autoimmune hypoglycemia due to autoantibodies to insulin, autoantibodies to the insulin receptor, or autoantibodies that are stimulatory to pancreatic β-cells, macrophage differentiation which leads to the formation of atherosclerotic plaques, inflammatory response, carcinogenesis, hyperplasia, adipocyte gene expression, adipocyte differentiation, reduction in the pancreatic β-cell mass, insulin secretion, tissue sensitivity to insulin, liposarcoma cell growth, polycystic ovarian disease, chronic anovulation, hyperandrogenism, progesterone production, steroidogenesis, redox potential and oxidative stress in cells, nitric oxide synthase (NOS) production, increased gamma glutamyl transpeptidase, catalase, plasma triglycerides, HDL, and LDL cholesterol levels, and the like.

In addition, the selective VPAC2 receptor peptide agonists of the invention may be used for treatment of asthma (Bolin, et al., Biopolymer 37:57-66 (1995); U.S. Pat. No. 5,677,419; showing that polypeptide R3PO is active in relaxing guinea pig tracheal smooth muscle); hypotension induction (VIP induces hypotension, tachycardia, and facial flushing in asthmatic patients (Morice, et al., Peptides 7:279-280 (1986); Morice, et al., Lancet 2:1225-1227 (1983)); male reproduction problems (Siow, et al., Arch. Androl. 43(1):67-71 (1999)); as an anti-apoptosis/neuroprotective agent (Brenneman, et al., Ann. N.Y. Acad. Sci. 865:207-12 (1998)); cardioprotection during ischemic events (Kalfin, et al., J. Pharmacol. Exp. Ther. 1268(2):952-8 (1994); Das, et al., Ann. N.Y. Acad. Sci. 865:297-308 (1998)), manipulation of the circadian clock and its associated disorders (Harmar, et al., Cell 109:497-508 (2002); Shen, et al., Proc. Natl. Acad. Sci. 97:11575-80, (2000)), and as an anti-ulcer agent (Tuncel, et al., Ann. N.Y. Acad. Sci. 865:309-22, (1998)).

An “effective amount” of a selective PEGylated VPAC2 receptor peptide agonist is the quantity that results in a desired therapeutic and/or prophylactic effect without causing unacceptable side effects when administered to a subject in need of VPAC2 receptor stimulation. A “desired therapeutic effect” includes one or more of the following: 1) an amelioration of the symptom(s) associated with the disease or condition; 2) a delay in the onset of symptoms associated with the disease or condition; 3) increased longevity compared with the absence of the treatment; and 4) greater quality of life compared with the absence of the treatment. For example, an “effective amount” of a VPAC2 agonist for the treatment of NIDDM is the quantity that would result in greater control of blood glucose concentration than in the absence of treatment, thereby resulting in a delay in the onset of diabetic complications such as retinopathy, neuropathy, or kidney disease. An “effective amount” of a selective PEGylated VPAC2 receptor peptide agonist for the prevention of NIDDM is the quantity that would delay, compared with the absence of treatment, the onset of elevated blood glucose levels that require treatment with anti-hypoglycemic drugs such as sulfonylureas, thiazolidinediones, insulin, and/or bisguanidines.

An “effective amount” of the selective PEGylated VPAC2 receptor peptide agonist administered to a subject will also depend on the type and severity of the disease and on the characteristics of the subject, such as general health, age, sex, body weight and tolerance to drugs. The dose of selective PEGylated VPAC2 peptide receptor agonist effective to normalize a patient's blood glucose will depend on a number of factors, among which are included, without limitation, the subject's sex, weight and age, the severity of inability to regulate blood glucose, the route of administration and bioavailability, the pharmacokinetic profile of the peptide, the potency, and the formulation.

A typical dose range for the selective PEGylated VPAC2 receptor peptide agonists of the present invention will range from about 1 μg per day to about 5000 μg per day. Preferably, the dose ranges from about 1 μg per day to about 2500 μg per day, more preferably from about 1 μg per day to about 1000 μg per day. Even more preferably, the dose ranges from about 5 μg per day to about 100 μg per day. A further preferred dose range is from about 10 μg per day to about 50 μg per day. Most preferably, the dose is about 20 μg per day.

A “subject” is a mammal, preferably a human, but can also be an animal, e.g., companion animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, sheep, pigs, horses, and the like) and laboratory animals (e.g., rats, mice, guinea pigs, and the like).

The selective VPAC2 receptor peptide agonists of the present invention can be prepared by using standard methods of solid-phase peptide synthesis techniques. Peptide synthesizers are commercially available from, for example, Rainin-PTI Symphony Peptide Synthesizer (Tucson, Ariz.). Reagents for solid phase synthesis are commercially available, for example, from Glycopep (Chicago, Ill.). Solid phase peptide synthesizers can be used according to manufacturers instructions for blocking interfering groups, protecting the amino acid to be reacted, coupling, decoupling, and capping of unreacted amino acids.

Typically, an α-N-protected amino acid and the N-terminal amino acid on the growing peptide chain on a resin is coupled at room temperature in an inert solvent such as dimethylformamide, N-methylpyrrolidone or methylene chloride in the presence of coupling agents such as dicyclohexylcarbodiimide and 1-hydroxybenzotriazole and a base such as diisopropylethylamine. The α-N-protecting group is removed from the resulting peptide resin using a reagent such as trifluoroacetic acid or piperidine, and the coupling reaction repeated with the next desired N-protected amino acid to be added to the peptide chain. Suitable amine protecting groups are well known in the art and are described, for example, in Green and Wuts, “Protecting Groups in Organic Synthesis”, John Wiley and Sons, 1991. Examples include t-butyloxycarbonyl (tBoc) and fluorenylmethoxycarbonyl (Fmoc).

The selective VPAC2 receptor peptide agonists are also synthesized using standard automated solid-phase synthesis protocols using t-butoxycarbonyl- or fluorenylmethoxycarbonyl-alpha-amino acids with appropriate side-chain protection. After completion of synthesis, peptides are cleaved from the solid-phase support with simultaneous side-chain deprotection using standard hydrogen fluoride methods or trifluoroacetic acid (TFA). Crude peptides are then further purified using Reversed-Phase Chromatography on Vydac C18 columns using acetonitrile gradients in 0.1% trifluoroacetic acid (TFA). To remove acetonitrile, peptides are lyophilized from a solution containing 0.1% TFA, acetonitrile and water. Purity can be verified by analytical reversed phase chromatography. Identity of peptides can be verified by mass spectrometry. Peptides can be solubilized in aqueous buffers at neutral pH.

The peptide agonists of the present invention may also be made by recombinant methods known in the art using both eukaryotic and prokaryotic cellular hosts.

Once a peptide for use in the present invention is prepared and purified, it is modified by covalently linking at least one PEG molecule to Cys or Lys residues, to K(W) or K(CO(CH₂)₂SH), or to the carboxy-terminal amino acid. A wide variety of methods have been described in the art to produce peptides covalently conjugated to PEG and the specific method used for the present invention is not intended to be limiting (for review article see, Roberts, M. et al. Advanced Drug Delivery Reviews, 54:459-476, 2002).

An example of a PEG molecule which may be used is methoxy-PEG2-MAL-40K, a bifurcated PEG maleimide (Nektar, Huntsville, Ala.). Other examples include, but are not limited to bulk mPEG-SBA-20K (Nektar) and mPEG2-ALD-40K (Nektar).

Carboxy-terminal attachment of PEG may be attached via enzymatic coupling using recombinant VPAC2 receptor peptide agonist as a precursor or alternative methods known in the art and described, for example, in U.S. Pat. No. 4,343,898 or Intl. J. Pept. & Prot. Res. 43:127-38 (1994).

One method for preparing the PEGylated VPAC2 receptor peptide agonists of the present invention involves the use of PEG-maleimide to directly attach PEG to a thiol group of the peptide. The introduction of a thiol functionality can be achieved by adding or inserting a Cys or hC residue onto or into the peptide at positions described above. A thiol functionality can also be introduced onto the side-chain of the peptide (e.g. acylation of lysine ε-amino group by a thiol-containing acid, such as mercaptopropionic acid). A PEGylation process of the present invention utilizes Michael addition to form a stable thioether linker. The reaction is highly specific and takes place under mild conditions in the presence of other functional groups. PEG maleimide has been used as a reactive polymer for preparing well-defined, bioactive PEG-protein conjugates. It is preferable that the procedure uses a molar excess, preferably from 1 to 10 molar excess, of a thiol-containing VPAC2 receptor peptide agonist relative to PEG maleimide to drive the reaction to completion. The reactions are preferably performed between pH 4.0 and 9.0 at room temperature for 10 minutes to 40 hours. The excess of unPEGylated thiol-containing peptide is readily separated from the PEGylated product by conventional separation methods. The PEGylated VPAC2 receptor peptide agonist is preferably isolated using reverse-phase HPLC or size exclusion chromatography. Specific conditions required for PEGylation of VPAC2 receptor peptide agonists are set forth in Example 7. Cysteine PEGylation may be performed using PEG maleimide or bifurcated PEG maleimide.

An alternative method for preparing the PEGylated VPAC2 receptor peptide agonists of the invention, involves PEGylating a lysine residue using a PEG-succinimidyl derivative. In order to achieve site specific PEGylation, the Lys residues which are not used for PEGylation are substituted for Arg residues.

Another approach for PEGylation is via Pictet-Spengler reaction. A Trp residue with its free amine is needed to incorporate the PEG molecule onto VPAC2 receptor selective peptide. One approach to achieve this is to site specifically introduce a Trp residue onto the amine of a Lys sidechain via an amide bond during the solid phase synthesis (see Example 9).

Various preferred features and embodiments of the present invention will now be described with reference to the following non-limiting examples:

EXAMPLE 1 Preparation of the Selective VPAC2 Receptor Peptide Agonists by Solid Phase t-Boc Chemistry

Selective VPAC2 receptor peptide agonists may be prepared using the following method and then PEGylating using one of the methods described in Examples 7, 8 and 9.

Approximately 0.5-0.6 grams (0.38-0.45 mmole) Boc Pro-MBHA resin is placed in a standard 60 mL reaction vessel. Double couplings are run on an Applied Biosystems ABI430A peptide synthesizer. The following side-chain protected amino acids (2 mmole cartridges of Boc amino acids) are obtained from Midwest Biotech (Fishers, Ind.) and are used in the synthesis:

Arg-Tosyl (TOS), Asp-δ-cyclohexyl ester(OcHx), Glu-δ-cyclohexyl ester (OcHx), His-benzyloxymethyl(BOM), Lys-2-chlorobenzyloxycarbonyl (2Cl-Z), Ser-O-benzyl ether (OBzl), Thr-O-benzyl ether (OBzl), Trp-formyl (CHO) and Tyr-2-bromobenzyloxycarbonyl (2Br-Z) and Boc Gly PAM resin. Trifluoroacetic acid (TFA), di-isopropylethylamine (DIEA), 0.5 M hydroxybenzotriazole (HOBt) in DMF and 0.5 M dicyclohexylcarbodiimide (DCC) in dichloromethane are purchased from PE-Applied Biosystems (Foster City, Calif.). Dimethylformamide (DMF-Burdick and Jackson) and dichloromethane (DCM-Mallinkrodt) is purchased from Mays Chemical Co. (Indianapolis, Ind.).

Standard double couplings are run using either symmetric anhydride or HOBt esters, both formed using DCC. At the completion of the syntheses, the N-terminal Boc group is removed and the peptidyl resins are treated with 20% piperidine in DMF to deformylate the Trp side chain if Trp is present in the sequence. For the N-terminal acylation, four-fold excess of symmetric anhydride of the corresponding acid is added onto the peptide resin. The symmetric anhydride is prepared by diisopropylcarbodiimde (DIC) activation in DCM. The reaction is allowed to proceed for 4 hours and monitored by ninhydrin test. After washing with DCM, the resins are transferred to a TEFLON reaction vessel and are dried in vacuo.

Cleavages are done by attaching the reaction vessels to a BF (hydrofluoric acid) apparatus (Penninsula Laboratories). 1 mL m-cresol per gram/resin is added and 10 mL HF (purchased from AGA, Indianapolis, Ind.) is condensed into the pre-cooled vessel. 1 mL DMS per gram resin is added when methionine is present. The reactions are stirred one hour in an ice bath. The HF is removed in vacuo. The residues are suspended in ethyl ether. The solids are filtered and are washed with ether. Each peptide is extracted into aqueous acetic acid and either is freeze dried or is loaded directly onto a reverse-phase column.

Purifications are run on a 2.2×25 cm VYDAC C18 column in buffer A (0.1% Trifluoroacteic acid in water, B: 0.1% TFA in acetonitrile). A gradient of 20% to 90% B is run on an HPLC (Waters) over 120 minutes at 10 mL/minute while monitoring the UV at 280 nm (4.0 A) and collecting one minute fractions. Appropriate fractions are combined, frozen and lyophilized. Dried products are analyzed by HPLC (0.46×15 cm METASIL AQ C18) and MALDI mass spectrometry.

EXAMPLE 2 Preparation of the Selective VPAC2 Receptor Peptide Agonists by Solid Phase FMoc Chemistry

Selective VPAC2 receptor peptide agonists may be prepared using the following method and then PEGylating using one of the methods described in Examples 7, 8 and 9.

Approximately 114 mg (50 mMole) FMOC-Rink amide resin (purchased from GlycoPep, Chicago, Ill.) is placed in each reaction vessel. The synthesis is conducted on a Rainin Symphony Peptide Synthesizer. Analogs with a C-terminal amide are prepared using 75 mg (50 μmole) Rink Amide AM resin (Rapp Polymere. Tuebingen, Germany).

The following FMOC amino acids are purchased from GlycoPep (Chicago, Ill.), and NovaBiochem (La Jolla, Calif.): Arg-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), Asn-trityl (Trt), Asp-β-t-Butyl ester (tBu), Glu-δ-t-butyl ester (tBu), Gln-trityl (Trt), His-trityl (Trt), Lys-t-butyloxycarbonyl (Boc), Ser-t-butyl ether (OtBu), Thr-t-butyl ether (OtBu), Trp-t-butyloxycarbonyl (Boc), Tyr-t-butyl ether (OtBu).

Solvents dimethylformamide (DMF-Burdick and Jackson), N-methylpyrrolidone (NMP-Burdick and Jackson), dichloromethane (DCM-Mallinkrodt) are purchased from Mays Chemical Co. (Indianapolis, Ind.).

Hydroxybenzotrizole (HOBt), di-isopropylcarbodiimde (DIC), di-isopropylethylamine (DEA), and piperidine (Pip) are purchased from Aldrich Chemical Co (Milwaukee, Wis.).

All amino acids are dissolved in 0.3 M in DMF. Three hour DIC/HOBt activated couplings are run after 20 minutes deprotection using 20% Piperidine/DMF. Each resin is washed with DMF after deprotections and couplings. After the last coupling and deprotection, the peptidyl resins are washed with DCM and are dried in vacuo in the reaction vessel. For the N-terminal acylation, four-fold excess of symmetric anhydride of the corresponding acid is added onto the peptide resin. The symmetric anhydride is prepared by diisopropylcarbodiimde (DIC) activation in DCM. The reaction is allowed to proceed for 4 hours and monitored by ninhydrin test. The peptide resin is then washed with DCM and dried in vacuo.

The cleavage reaction is mixed for 2 hours with a cleavage cocktail consisting of 0.2 mL thioanisole, 0.2 mL methanol, 0.4 mL triisopropylsilane, per 10 mL trifluoroacetic acid (TFA), all purchased from Aldrich Chemical Co., Milwaukee, Wis. If Cys is present in the sequence, 2% of ethanedithiol is added. The TFA filtrates are added to 40 mL ethyl ether. The precipitants are centrifuged 2 minutes at 2000 rpm. The supernatants are decanted. The pellets are resuspended in 40 mL ether, re-centrifuged, re-decanted, dried under nitrogen and then in vacuo.

0.3-0.6 mg of each product is dissolved in 1 mL 0.1% TFA/acetonitrile (ACN), with 20 μL being analyzed on HPLC [0.46×15 cm METASIL AQ C18, 1 mL/min, 45° C., 214 nM (0.2 A), A=0.1% TFA, B=0.1% TFA/50% ACN. Gradient=50% B to 90% B over 30 minutes].

Purifications are run on a 2.2×25 cm VYDAC C18 column in buffer A (0.1% trifluoroacteic acid in water, B: 0.1% TFA in acetonitrile). A gradient of 20% to 90% B is run on an HPLC (Waters) over 120 minutes at 10 mL/minute while monitoring the UV at 280 nm (4.0 A) and collecting 1 minute fractions. Appropriate fractions are combined, frozen and lyophilized. Dried products are analyzed by HPLC (0.46×15 cm METASIL AQ C18) and MALDI mass spectrometry.

EXAMPLE 3 In Vitro Potency

DiscoveRx: A CHO—S cell line stably expressing human PEGylated VPAC2 receptor in a 96-well microtiter plate is seeded with 50,000 cells/well the day before the assay. The cells are allowed to attach for 24 hours in 200 μL culture medium. On the day of the experiment, the medium is removed. Also, the cells are washed twice. The cells are incubated in assay buffer plus BMX for 15 minutes at room temperature. Afterwards, the stimuli are added and are dissolved in assay buffer. The stimuli are present for 30 minutes. Then, the assay buffer is gently removed. The cell lysis reagent of the DiscoveRx cAMP kit is added. Thereafter, the standard protocol for developing the cAMP signal as described by the manufacturer is used (DiscoveRx Inc., USA). EC₅₀ values for cAMP generation are calculated from the raw signal or are based on absolute cAMP levels as determined by a standard curve performed on each plate. In the case of VPAC1 and PAC1 receptors, CHO—PO cells are transiently transfected with human VPAC1 or PAC1 receptor DNA using commercially available transfection reagents (Lipofectamine from Invitrogen). The cells are seeded at a density of 10,000/well in a 96-well plate and are allowed to grow for 3 days in 200 mL culture medium. At day 3, the assay described above for the PEGylated VPAC2 receptor cell line is performed.

Results for each agonist are the mean of two independent runs. VPAC1 and PAC1 results are only generated using the DiscoveRx assay. The typically tested concentrations of peptide are: 1000, 300, 100, 10, 1, 0.3, 0.1, 0.01, 0.001, 0.0001 and 0 nM.

Alpha screen: Cells are washed in the culture flask once with PBS. Then, the cells are rinsed with enzyme free dissociation buffer. The dissociated cells are removed. The cells are then spun down and washed in stimulation buffer. For each data point, 50,000 cells suspended in stimulation buffer are used. To this buffer, Alpha screen acceptor beads are added along with the stimuli. This mixture is incubated for 60 minutes. Lysis buffer and Alpha screen donor beads are added and are incubated for 60 to 120 minutes. The Alpha screen signal (indicative of intracellular cAMP levels) is read in a suitable instrument (e.g. AlphaQuest from Perkin-Elmer). Steps including Alpha screen donor and acceptor beads are performed in reduced light. The EC₅₀ for cAMP generation is calculated from the raw signal or is based on absolute cAMP levels as determined by a standard curve performed on each plate.

Results for each agonist are, at minimum, from two analyses performed in a single run. For some agonists, the results are the mean of more than one run. The tested peptide concentrations are: 10000, 1000, 100, 10, 3, 1, 0.1, 0.01, 0.003, 0.001, 0.0001 and 0.00001 nM. The activity (EC₅₀ (nM)) for the human PEGylated VPAC2, VPAC1, and PAC1 receptors is reported in Table 1.

TABLE 1 Human Human Human VPAC2 VPAC2 VPAC1 Human PAC1 Receptor: Receptor: Receptor: Receptor: Agonist # DiscoveRx¹ Alpha Screen² DiscoverRx¹ DiscoverRx¹ VIP (SEQ 0.70 1.00 0.02 15.4 ID NO: 1) PACAP- 0.84 2.33 0.05 0.06 27 VPAC1 179.29 n.d. n.d. n.d. P1 P136 n.d. 8.68 n.d. n.d. ¹EC₅₀ (nM); Mean of two independent runs ²EC₅₀ (nM); Single result from two analyses performed in a single run Mean of separate results for the given assay n.d. = not determined

EXAMPLE 4 Selectivity

Binding assays: Membrane prepared from a stable VPAC2 cell line (see Example 3) or from cells transiently transfected with human VPAC1 or PAC1 are used. A filter binding assay is performed using 125I-labeled VIP for VPAC1 and VPAC2 and 125I-labeled PACAP-27 for PAC1 as the tracers. For this assay, the solutions and equipment include:

Presoak solution: 0.5% Polyethyleneamine in Aqua dest.

Buffer for flushing filter plates: 25 mM HEPES pH 7.4

Blocking buffer: 25 mM HEPES pH 7.4; 0.2% protease free BSA

Assay buffer: 25 mM HEPES pH 7.4; 0.5% protease free BSA

Dilution and assay plate: PS-Microplate, U form

Filtration Plate Multiscreen FB Opaque Plate; 1.0 μM Type B Glasfiber filter

In order to prepare the filter plates, the presoak solution is aspirated by vacuum filtration. The plates are flushed twice with 200 μL flush buffer. 200 μL blocking buffer is added to the filter plate. The filter plate is then incubated with 200 μL presoak solution for 1 hour at room temperature.

The assay plate is filled with 25 μL assay buffer, 25 μL membranes (2.5 μg) suspended in assay buffer, 25 μL compound (agonist) in assay buffer, and 25 μL tracer (about 40000 cpm) in assay buffer. The filled plate is incubated for 1 hour with shaking.

The transfer from assay plate to filter plate is conducted. The blocking buffer is aspirated by vacuum filtration and washed two times with flush buffer. 90 μL is transferred from the assay plate to the filter plate. The 90 μL transferred from assay plate is aspirated and washed three times with 200 μL flush buffer. The plastic support is removed. It is dried for 1 hour at 60° C. 30 μL Microscint is added. The count is performed.

The selectivity (IC₅₀) for human PEGylated VPAC2, VPAC1, and PAC1 is reported in Table 2.

TABLE 2 Human VPAC2 Human VPAC1 Human PAC1 Agonist # Receptor Binding Receptor Binding Receptor Binding VIP (SEQ ID 5.06 3.28 >25000 NO: 1) PACAP-27 2.76 3.63 9.1 P136 0.90 617.41 2257.3

Values given are the result of a single or the mean of two or more experiments

TABLE 3 In vitro potency using DiscoveRx (see Example 3). CHO-PO cells are transiently transfected with rat VPAC1 or VPAC2 receptor DNA. The activity (EC₅₀ (nm)) for these receptors is reported in the table below. Rat VPAC 2 Receptor Rat VPAC 1 Receptor Agonist # DiscoveRx DiscoveRx VIP 0.79 0.02 PACAP-27 n.d. 0.07 P136 n.d. 15.00

EXAMPLE 5 In Vivo Assay

Glucose lowering in diabetic ZDF rats. ZDF rats, 8-9 weeks old with fed glucose levels of approximately 300 mg/dl are used for this experiment. The animals are randomised into control (vehicle) and treatment group(s) on the day of the experiment and are conscious throughout the experiment. The compound is injected intravenously at the start of the experiment and blood samples are drawn from the tail vein immediately prior to compound injection and then 0.5, 1, 2, 3, 4 and 24 h after compound injection. The animals are deprived of food during the first 2 or 4 h of the experiment. The blood samples are collected in EDTA tubes, aprotinin added and immediately put on ice pending insulin and glucose analysis using standard methods.

TABLE 4 Glucose lowering in conscious food-deprived ZDF rats Time after P136 injection (μg/kg) Analyte 0 h 0.5 h 1 h 2 h 3 h 4 h 24 h Vehicle glucose 298 n.d. 332 253 219 215 366 (mg/dl) Vehicle insulin 17.1 n.d. 13.0 10.5 9.3 10.6 10.3 (ng/ml) 100 glucose 347 n.d. 333 280 212 207 400 (mg/dl) 100 insulin 19.1 n.d. 21.3 18.0 16.0 16.1 9.3 (ng/ml) Animals are given access to food after the 4 h timepoint

EXAMPLE 6 Rat Serum Stability Studies

In order to determine the stability of PEGylated VPAC2 receptor peptide agonists in rat serum, obtain CHO-VPAC2 cells clone #6 (96 well plates/50,000 cells/well and 1 day culture), PBS 1× (Gibco), the peptides for the analysis in a 100 μM stock solution, rat serum from a sacrificed normal Wistar rat, aprotinin, and a DiscoveRx assay kit. The rat serum is stored at 4° C. until use and is used within two weeks.

On Day 0, two 100 μL aliquots of 10 μM peptide in rat serum are prepared by adding 10 μL peptide stock to 90 μL rat serum for each aliquot. 250 kIU aprotinin/mL is added to one of these aliquots. The aliquot is stored with aprotinin at 4° C. The aliquot is stored without aprotinin at 37° C. The aliquots are incubated for 18 hours.

On Day 3, after incubation of the aliquots prepared on day 0 for 72 hours, an incubation buffer containing PBS+1.3 mM CaCl₂, 1.2 mM MgCl₂, 2 mM glucose, and 0.25 mM IBMX is prepared. A plate with 11 serial 5× dilutions of peptide for the 4° C. and 37° C. aliquot is prepared for each peptide studied. 2000 nM is used as the maximal concentration if the peptide has an EC₅₀ above 1 nM and 1000 nM as maximal concentration if the peptide has an EC₅₀ below 1 nM from the primary screen (see Example 3). The plate(s) are washed with cells twice in incubation buffer. The plates are allowed to hold 50 μL incubation media per well for 15 minutes. 50 μL solution per well is transferred to the cells from the plate prepared with 11 serial 5× dilutions of peptide for the 4° C. and 37° C. aliquot for each peptide studied, using the maximal concentrations that are indicated by the primary screen, in duplicate. This step dilutes the peptide concentration by a factor of two. The cells are incubated at room temperature for 30 minutes. The supernatant is removed. 40 μL/well of the DiscoveRx antibody/extraction buffer is added. The cells are incubated on shaker (300 rpm) for 1 hour. Normal procedure with the DiscoveRx kit is followed. cAMP standards are included in column 12. EC₅₀ values are determined from the cAMP assay data. The remaining amount of active peptide is estimated by the formula EC_(50, 4C)/EC_(50, 37C) for each condition.

TABLE 5 Rat Serum Stability Peptide (Estimated purity in % after 72 hours) P136 34.4

EXAMPLE 7 PEGylation of Selective VPAC2 Receptor Peptide Agonist Using Thiol-Based Chemistry

Usually a thiol function is introduced into or onto a selective VPAC2 receptor peptide agonist by adding a cysteine or a homocysteine or a thiol-containing moiety at either or both termini or by inserting a cysteine or a homocysteine or a thiol-containing moiety into the sequence. Thiol-containing VPAC2 receptor peptide agonists are reacted with 40 kDa polyethylene glycol-maleimide (PEG-maleimide) to produce derivatives with PEG covalently attached via a thioether bond.

PEGylation reactions are run under conditions that permit the formation of a thioether bond. Specifically, the pH of the solution ranges from about 4 to 9 and the thiol containing peptide concentrations range from 1 to 10 molar excess of PEG-maleimide. A PEG-maleimide which may be used is methoxy-PEG2-MAL-40K, a bifurcated PEG maleimide (Lot # PT-06D-01, Nektar, Huntsville, Ala.). The PEGylation reactions are normally run at room temperature for 10 minutes to 40 hours. The PEGylated VPAC2 receptor peptide agonist is then isolated using reverse-phase HPLC or size exclusion chromatography (SEC). PEGylated peptide analogues are characterized using analytical RP-HPLC, HPLC-SEC, SDS-PAGE, and/or MALDI Mass Spectrometry.

EXAMPLE 8 PEGylation via Acylation on the Sidechain of Lysine

In order to achieve site-specific PEGylation of selective VPAC2 receptor peptide agonists, all the Lys residues are changed into Arg residues except for the Lys residues where PEGylation is intended. A PEG molecule which may be used is mPEG-SBA-20K (Nektar, Lot #: PT-04E-11). The PEGylation reaction us preferably performed at room temperature for 2-3 hours. The protein is purified by preparative HPLC.

EXAMPLE 9 PEGylation via Pictet-Spengler Reaction

For PEGylation via Pictet-Spengler reaction to occur, a Trp residue with its free amine is needed to incorporate the PEG molecule onto the selective VPAC2 receptor peptide agonist. One approach to achieve this is to add a Lys residue onto the C-terminus of the peptide and then to couple a Trp residue onto the sidechain of Lys. The extensive SAR indicates that this modification does not change the properties of the parent peptide in terms of its in vitro potency and selectivity.

PEG with a functional aldehyde, for example mPEG2-ALD-40K (Nektar, Lot #: PT-6C-05), is used for the reaction. The site specific PEGylation involves the formation a tetracarboline ring between PEG and the peptide. PEGylation is conducted in glacial acetic acid at room temperature for 1 to 48 hours. A 1 to 10 molar excess of the PEG aldehyde is used in the reaction. After the removal of acetic acid, the PEGylated VPAC2 receptor peptide agonist is isolated by preparative RP-HPLC.

For the PEGylation of P118, CH₃—(CH₂)₄—CO—HSDAVFTDNYTRLRKQVAAKKYLQSIKNSRTSPPPK(W)-NH₂, (3.5 mg, 0.8 umol), 54 mg of mPEG2-ALD-40K (Nektar, Lot #: PT-6C-05) and 1 ml of glacial acetic acid are used. The P118 and the mPEG2-ALD-40K are dissolved in the acetic acid. The reaction is allowed to proceed for 18 hours. 40 mg of the PEGylated peptide (VPAC2-P136) is isolated by preparative RP-HPLC, characterised by SE-HPLC and tested for in-vitro activity.

Other modifications of the present invention will be apparent to those skilled in the art without departing from the scope of the invention. 

1-48. (canceled)
 49. A PEGylated VPAC2 receptor peptide agonist, comprising the amino acid sequence: (SEQ ID NO: 16) His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Xaa₉-Tyr-Thr-Arg- Leu-Xaa₁₄-Xaa₁₅-Xaa₁₆-Xa₁₇-Ala-Ala-Xaa₂₀-Lys-Tyr- Leu-Gln-Ser-Ile-Lys-Xaa₂₈

wherein: Xaa₉ is: Asn, or Gln; Xaa₁₄ is: Arg, or Leu; Xaa₁₅ is: Lys, Leu, or Aib; Xaa₁₆ is: Gln, Lys, or Ala; Xaa₁₇ is: Val, or Ala; Xaa₂₀ is: Lys, or Aib; and Xaa₂₈ is: Asn, or Gln; and a C-terminal extension, wherein the N-terminus of said C-terminal extension is linked to the C-terminus of said peptide of SEQ ID NO: 16, wherein said C-terminal extension comprises the amino acid sequence: (SEQ ID NO: 17) Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉

wherein: Xaa₁ is: Ser or absent; Xaa₂ is: Arg or absent; Xaa₃ is: Thr or absent; Xaa₄ is: Ser or absent; Xaa₅ is: Pro or absent; Xaa₆ is: Pro or absent; Xaa₇ is: Pro or absent; Xaa₈ is: Lys, K(W), Cys, or absent; and Xaa₉ is: K(E-C₁₆) or absent; provided that at least four of Xaa₁ to Xaa₉ of said C-terminal extension are present, and provided that if Xaa₁, Xaa₂, Xaa₃, Xaa₄, Xaa₅, Xaa₆, Xaa₇, or Xaa₈ is absent, the next amino acid present downstream is the next amino acid in SEQ ID NO: 17, and wherein said C-terminal amino acid is optionally amidated, and wherein the Cys residue, when present in said C-terminal extension, is covalently attached to a PEG molecule, or wherein at least one of the Lys residues in said VPAC2 receptor peptide agonist is covalently attached to a PEG molecule, or wherein K(W), when present in said C-terminal extension, is covalently attached to a PEG molecule, or wherein the carboxy-terminal amino acid of said VPAC2 receptor peptide agonist is covalently attached to a PEG molecule, or a combination of any one of the foregoing, or a pharmaceutically acceptable salt thereof.
 50. The PEGylated VPAC2 receptor peptide agonist according to claim 49, wherein said C-terminal extension is an amino acid sequence selected from the group consisting of SRTSPPP (SEQ ID NO: 9), SRTSPPP-NH₂ (SEQ ID NO: 10), SRTSPPPK(W) (SEQ ID NO: 23), SRTSPPPK(W)-NH₂ (SEQ ID NO: 24), SRTSPPPC (SEQ ID NO: 25), and SRTSPPPC-NH₂ (SEQ ID NO: 26).
 51. The PEGylated VPAC2 receptor peptide agonist according to claim 49, wherein said PEG molecule is branched.
 52. The PEGylated VPAC2 receptor peptide agonist according to claim 49, wherein said PEG molecule is linear.
 53. The PEGylated VPAC2 receptor peptide agonist according to claim 49, wherein said PEG molecule is 20,000, 40,000, or 60,000 daltons in molecular weight.
 54. The PEGylated VPAC2 receptor peptide agonist according to claim 49, wherein two PEG molecules are present, and each of said PEG molecules is 20,000 daltons in molecular weight.
 55. The PEGylated VPAC2 receptor peptide agonist according to claim 49, further comprising an N-terminal modification, wherein said N-terminal modification is the addition of a group selected from the group consisting of acetyl, hexanoyl, propionyl, 3-phenylpropionyl, and benzoyl.
 56. The PEGylated VPAC2 receptor peptide agonist according to claim 49, comprising the amino acid sequence: (SEQ ID NO: 44) hexanoyl-HSDAVFTDNYTRLRKQVAAKKYLQSIKNSRTSPPPK (WPEG40K)-NH₂.


57. (canceled)
 58. A method of treating non-insulin-dependent diabetes or insulin-dependent diabetes in a mammal in need thereof, comprising administering to said mammal an effective amount of a PEGylated VPAC2 receptor peptide agonist according to claim
 49. 59. The method of claim 58, wherein said mammal is a human. 